Use of cellular extracts for skin rejuvenation

ABSTRACT

The invention describes methods and agents for improving cosmetic appearance, for promoting, improving or restoring health of cells and tissues, preferably skin, and more preferably, for restoring aged or damaged skin to a healthy appearance. In preferred embodiments, the methods and agents comprise active extracts produced from fish eggs. The invention further provides processes for making active fish egg extracts.

This application is a continuation of pending U.S. patent applicationSer. No. 14/048,586, which will issue on Dec. 30, 2014 as U.S. Pat. No.8,920,848, which is a continuation of pending U.S. patent applicationSer. No. 13/101,445, filed May 5, 2011, now allowed as U.S. Pat. No.8,557,295 which issued on Oct. 15, 2013, which claims the benefit ofexpired U.S. Provisional Patent Application No. 61/332,047, filed May 6,2010; and is a continuation-in-part of pending U.S. patent applicationSer. No. 12/437,100, filed May 7, 2009, now allowed as U.S. Pat. No.8,877,253, which claims the benefit of expired U.S. Provisional PatentApplication No. 61/051,931, filed May 9, 2008 and expired U.S.Provisional Patent Application No. 61/120,146, filed Dec. 5, 2008; andis a continuation in part of U.S. patent application Ser. No.11/801,778, filed May 11, 2007, allowed as U.S. Pat. No. 8,075,920,which claims the benefit expired U.S. Provisional Patent Application No.60/799,560, filed May 11, 2006, of all of which are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the use of compositions comprisingdifferentiable cells, egg cellular extracts or differentiable cellcellular extracts to prevent deterioration, damage and malfunction ofcells and tissues, and to promote, improve and exceed cellular functionin order to promote, improve and exceed appearance, vitality and healthof cells and tissues, especially skin.

BACKGROUND OF THE INVENTION

Skin is the first barrier we have against outside aggressions, andcarries out both physical and chemical defenses. Vitamin D is producedin the epidermis under the effects of solar radiation. This vitamin isnecessary for calcium to be absorbed in the intestine and then fixed onthe bones, which enables the development and growth of the human body.However, excessive sun exposure leads to skin damage and potentiallycancer. In addition, skin cells may become damaged by physical means,i.e., wounded, or damaged due to age. In addition, aging decreases theactivity of skin cells, especially fibroblasts, and the secretion ofcollagen from fibroblast. Thus, there is a need to identify compositionsand methods for managing and improving skin health and preventing andtreating skin conditions, and diseases, and maintaining normal skinappearance and restoring aged skin to a youthful appearance.

SUMMARY OF THE INVENTION

The invention relates to improving visible parts of a personcontributing to cosmetic appearance directly or indirectly, includingbut not limited to skin, and to improve health and damage of cells andtissues preferably skin, and more preferably restoring aged skin to ayouthful appearance. In some embodiments, the invention relates tocompositions of cells, cell or egg extracts, and extract componentswhich can induce de-differentiation, including but not limited topurified or synthetic nucleic acid sequences, polypeptides, or naturalproducts contained in the extracts. In some embodiments, the cells aredifferentiable cells, preferably stem cells. In some embodiments, thecompositions are used in a method that comprises application of thecompositions to skin and/or wounds after removal the outer surfacelayers. In some embodiments, the invention relates to a method ofde-differentiation of cells and/or de-differentiation followed byre-differentiation. In some embodiments, the invention relates tomanaging, preventing, and treating skin diseases. In some embodiments,the invention relates to a composition comprising i) a cellularcomponent comprising differentiable cells, differentiable cell cellularextracts, egg cellular extracts or components of differentiable cellextracts or egg cellular extracts or combinations thereof and ii)lipids. In further embodiments, the composition further comprisespurified or synthetic nucleic acid sequences, proteins, epigeneticinhibitors, or natural products contained in the extracts orcombinations thereof. In further embodiments, the differentiable cellsare embryonic stem cells, embryonic germ cells, or adult stem cells. Thepresent invention is not limited to the use of any particular cellularextract or fraction. Indeed, the use of a variety of cellular extractsand fractions is contemplated, including, but not limited to,cytoplasmic extracts and fractions, nuclear extracts and fractions,water soluble extracts and fractions, and extracts and fractionsprepared from cellular extracts by affinity chromatography, gradientcentrifugation, HPLC, size exclusion chromatography and the like.

In some embodiments, the invention provides methods and the compositionsfind use for prevention of deterioration, damage and malfunction ofcells and tissues, and to promote, improve and exceed cellular functionin order to promote, improve and exceed appearance, vitality and healthof cells and tissues.

In some embodiments, the lipid component is from a source other than thesource of the extract, e.g., a purified lipid from a different source,either natural or synthetic. In further embodiments, the lipid componentis derived from egg from fish, shrimp, sea urchin or frog and/or fishroe. In further embodiments, the lipid component contains cholesterol,fatty acids, and ceramides. In some embodiments, the lipid component isfrom a source different than the cellular component. In furtherembodiment, the composition contains keratin or flaggrin. In furtherembodiments, the composition further comprises glutamine, antiinfectiveagents, antioxidants and/or nicotinamide. In further embodiments, theantioxidant is vitamin E, A, or C or combinations thereof.

In some embodiments the invention provides a kit for improving theappearance of a scar comprising two compositions, wherein the firstcomposition dissolves scar tissue and comprises collagen dissolvingagents and the second composition improves wound healing and comprises acellular component selected from the group consisting of differentiablecells, differentiable cell cellular extracts and an egg cellular extractor combinations thereof, lipids, proteins, and water. In furtherembodiments, the first composition further comprises an antisepticcompound, an antibacterial compound, an anti-inflammatory compound, animmunomodulator, a protease, or an analgesics or combinations thereof.In further embodiments, the second composition further comprises naturalvernix, vernix extracts, vernix made from synthetic substances, andcomponents of vernix extracts. In further embodiments the lipidcomponent comprises squalene, aliphatic waxes, sterol esters, diolesters, triglycerides, free sterols, or combinations thereof. In furtherembodiments, the lipids and/or proteins are derived from eggs from fish,shrimp, sea urchin or frog and/or fish roe. In further embodiments, thelipid fraction contains cholesterol, fatty acids, or ceramides or acombination thereof. In some embodiments, the lipid component is from adifferent source than the cellular component. In further embodiments,the composition further comprises glutamine, antiinfective agents,antioxidants and/or nicotinamide.

In some embodiments, the invention provides methods for improving theappearance of a skin comprising: i) removing skin tissue by chemicals, alaser, or physical force and ii) applying a composition that improveswound healing comprising differentiable cells, differentiable cell oregg cellular extracts, components of differentiable cell extracts,lipids, proteins, and/or water. In further embodiments, improving theappearance of skin includes improving the appearance of a scar orimproving the appearance of skin with wrinkles. In further embodiments,the differentiable cells are embryonic stem cells, embryonic germ cells,or adult stem cells. In further embodiments, the composition furthercomprises natural vernix, vernix extracts, vernix made from syntheticsubstances, and components of vernix extracts.

In additional embodiments, the invention provides methods for thetopical administration of differentiable cells, egg or differentiablecell cellular extracts, components of cell extracts comprising:providing a composition comprising a cellular component comprisingdifferentiable cells, egg or differentiable cell cellular extracts,components of cell extracts and a subject having skin and applying theextracts to the skin of the subject. In further embodiments, the egg ordifferentiable cellular extracts or components of cell extracts areeffective as a nutrient to a cell of the skin. In further embodiments,the composition is a water-based gel. In further embodiments, thewater-based gel comprises a compound selected from the group consistingof hyaluronic acid and chitosan. In further embodiments the compositionis a component on a wound dressing. In further embodiments thecomposition is a component in a spray composition. In furtherembodiments the spray composition is an aerosol. In further embodiments,the spray composition dries on the skin. In further embodiments, thespray composition comprises gel-forming components. In some embodiments,the composition further comprises a lipid component as described above.

In some embodiments, the invention provides a wound healing dressingcomprising a composition comprising differentiable cells, egg ordifferentiable cell cellular extracts, and components of cell extracts.

In additional embodiments, the invention provides methods for thetopical administration of differentiable cells, cell extracts,components of cell extracts comprising: i) providing a) a compositioncontaining differentiable cells, differentiable cell or egg cellularextracts, components of cell extracts, b) a subject having a wound inskin and c) wound dressing ii) applying the differentiable cells, cellextracts, components of cell extracts to the wound; and iii) coveringthe wound with the wound dressing. In further embodiments, the wounddressing is non-occlusive. In further embodiments, the wound dressing isplaster. In further embodiments, the wound dressing comprises: i) awaterproof layer; ii) a nutrient gel layer comprising differentiablecells, cell extracts, and components of cell extracts. In furtherembodiments, the waterproof layer is a polymeric (i.e., plastic)membrane that can be glued onto skin. In further embodiments, thenutrient gel layer comprises antibacterial agents and collagenmodulating substances. In further embodiments, the nutrient gel layerimproves the speed of wound healing.

In some embodiments, the invention provides methods for the topicaladministration of differentiable cells, egg or differentiable cellcellular extracts, or components of cell extracts comprising: i)providing a) a subject having 1) a wound in skin and 2) a tissuecomprising specialized cells b) wound dressing; ii) harvesting thespecialized cells from the tissue; iii) culturing the specialized cellsunder conditions such that a composition comprising the culturedspecialized differentiable cells, cell extracts, or components of cellextracts is formed; iii) applying the composition to the wound; and iv)covering the wound with the wound dressing. In further embodiments, thespecialized cells selected from the group consisting of a bulgehair-follicle stem cell, an embryonic stem, or germ stem cell. Infurther embodiments, the composition is a fluid suspension ofspecialized cells. In further embodiments, the composition is a plaster.In further embodiments, the composition is placed on a membrane with anutrient gel layer prior to applying the composition to the wound. Infurther embodiments, the membrane is polymeric (i.e., plastic)functioning as an occlusive wound dressing when applied to the skin. Infurther embodiments, the wound dressing is a commercial band-aid. Infurther embodiments, prior to applying the composition a step of burningskin is performed, freezing skin is performed, and/or sanding skin isperformed. In further embodiments, prior to applying the composition atransport vehicle which penetrate intact skin is applied to thecomposition or skin comprising a phospholipids, palmitylmyristrates,DMSO, polymer or chitosan suspensions or matrix, liposomes and/or trojanpeptides, chariot peptides, small elastic vesicles (Van den Bergh etal., 1999), microspheres, nanoparticles, preloaded spherical beads, uni-and/or multilamellar vesicles, retinol molecular film, poly acrylonitrile, beta-glucan (Redmond, Int. Journ. Cosmetic Science 2005),propylene glycol, butylenes glycol, polyethylene glycol, olive oil,dimethyl isosorbate, dimethylformamide, methyl salicylate, long chainoleic acids.

In some embodiments, the invention provides compositions for stimulatingcells such as fibroblasts and keratinocytes comprising an effectiveamount of a purified cytoplasmic fraction of an embryonic stem cell,progenitor cell, somatic cell or eggs from animals, including but notlimited to primates, rodents, fish, shrimp, sea urchin and/or frog egg.In further embodiments, the composition further comprises fats, proteinsand/or natural products. In further embodiments, the composition furthercomprises an herbal substance. In further embodiments, the herbalsubstance is aloe vera. In further embodiments, the composition furthercomprises seed extracts. In further embodiments, the seed extracts areobtained from wheat, corn, rice, or avocado. In further embodiments, thecomposition further comprises a plant oil. In further embodiments, thecomposition further comprises a fungal substance. In furtherembodiments, the fungal substance is nepal fungus. In furtherembodiments, the composition further comprises fish, shrimp, sea urchin,or frog egg extracts, or components of these egg extracts. In furtherembodiments the components of egg extracts are glycosylation breakersand inhibitors. In further embodiments, the components of egg extractsare glycosylation breakers and inhibitors are aminoguanidine, carnosine,and fex pyridoxamine.

In additional embodiments, the invention provides methods of woundhealing comprising providing a subject having a wound and a compositioncomprising differentiable cells, differentiable cell or egg cellularextracts, egg extracts, components of cell extracts or egg extracts andapplying the composition to the wound under conditions such that thewound is healed. In further embodiments, the composition furthercomprises a collagen dissolving agent. In further embodiments, thecollagen dissolving agent is an acid. In further embodiments, thecomposition further comprises a fruit acid. In further embodiments thecomposition is a cream. In further embodiments, the wound is an openwound and applying the composition topically. In preferred embodiments,the method further comprises providing a support matrix wherein, thesupport matrix comprises the composition. In further embodiments, thesupport matrix is a fabric or plastic wound dressing.

In some embodiments, the invention provides methods of skin regenerationcomprising providing a subject having a wound and a compositioncomprising differentiable cells, differentiable cell or egg cellularextracts, or components of cell extracts or egg extracts and applyingthe composition to the wound under conditions that such skin isregenerated. In further embodiments the composition is a cream. Infurther embodiments, the wound is an open wound and the composition isapplied topically.

In additional embodiments, the invention relates to a method of skinrejuvenation comprising providing a subject having an uneven skin and acomposition comprising differentiable cells, differentiable cell or eggcellular extracts, egg extracts, or a component of a cellular extractand applying the composition to the uneven skin under conditions thatsuch skin is rejuvenated. In some embodiments, the component of a cellextract is a nucleic acid sequence or the component of a cell extract isa peptide or combinations thereof. In some embodiments, the uneven skinis a result of a scar or wrinkles. In further embodiments, thecomposition is in a cream. In further embodiments the cream furthercomprises permeabilizing agents. In further embodiments, thepermeabilizing agent is a non-toxic agent, DMSO or chitosan, chitosanpolymer, or trypsin. In further embodiments, the permeabilizing agent isliposomes or alginate beads. In further embodiments, the liposomes oralginate beads comprise a peptide or a nucleic acid sequence of a cellextract or growth factor or a combination thereof. In furtherembodiments, the liposome comprises nucleic acid sequence of cellextracts or egg extracts generated by electroporation. In furtherembodiments, the composition comprises a fusion trojan peptidecomprising a peptide of the cell extract. In further embodiments, thecomposition is applied topically. In additional embodiments, the methodfurther comprises the step of applying the composition is executed afterapplying a chemical, laser, or physical force to the uneven skin underconditions that an outer lay of cells of the uneven skin are removed. Infurther embodiments, the composition further comprises an antisepticcompound, an antibacterial compound, an anti-inflammatory compound, animmunomodulator, a protease, or an analgesic compound or combinationsthereof.

In some embodiments, the invention relates to a composition comprising:a lipid; a composition of plant seed components; an antioxidant; apurified or synthetic protein, or a purified or synthetic naturalproduct contained in a cellular extract; a stabilizing component;autologous fat derived from adipose tissue of a subject.

In additional embodiments, the invention provides methods of improving askin graft comprising grafting skin or skin substitute and applying acomposition comprising: differentiable cells, differentiable cell or eggcellular extracts, egg extracts; components of cell extracts or eggextracts; a purified or synthetic nucleic acid sequence, a purified orsynthetic protein, or a purified or synthetic natural product contain incell extracts, egg extracts; or combinations thereof.

In some embodiments, the invention provides methods for managing,treating, and/or preventing scarring, abnormal scars, abnormal woundhealing, widened scar, hypertrophied scar, keloid, keloid scar,wound-healing complications, cicatrix, and/or scar hypertrophy byadministering in a prophylactic or non-prophylactic manner thecompositions disclosed herein. In further embodiments, the inventionprovides methods for primary healing, wound closure, secondary healing,epithelialization, re-epithelialization, tertiary wound closure, delayedprimary closure, debridement, and suture using the compositionsdescribed herein. In other embodiments, the compositions describedherein are used to increase or decrease at the site of administration toa subject, inflammatory phase, proliferative phase, maturational phase,hemostasis, inflammation, collagen, clotting, thromboxane A2,prostaglandin 2a, prostaglandin 2-alpha, vasoconstrictor, hemorrhage,vasodilatation, histamine, platelet, chemokine, epidermal growth factor,fibronectin, fibrinogen, histamine, platelet derived growth factor,serotonin, von Willebrand factor, clot formation, plateletdegranulation, complement cascade, neutrophil, leukocyte, macrophage,monocyte, collagenase, interleukin, tumor necrosis factor, fibroblasts,transforming growth factor, keratinocyte, angiogenesis, granulationtissue formation, collagen deposition, and insulin-like growth factor.

In some embodiments, the invention provides compositions comprisingdifferentiable cells, preferably embryonic stem cells or precursorcells. In further embodiments, the compositions comprise the extracts ofdifferentiable cells, preferably embryonic stem cells or precursorcells. In additional embodiments, the compositions contain components ofextracts from differentiable cells, preferably embryonic stem cells orprecursor cells.

In some embodiments, the invention provides compositions containingdifferentiable cells, preferably embryonic stem cells or precursorcells, the extracts of differentiable cells, preferably embryonic stemcells or precursor cells, components of extracts from differentiablecells, and/or natural vernix and/or vernix extracts and/or vernixcomponents of vernix extracts that partially or totally synthetic.

In some embodiments, the invention provides methods for the topicaladministration of egg cellular extracts or differentiable cell cellularextracts comprising: providing a composition containing egg cellularextracts or differentiable cell cellular extracts and a subject havingskin and applying the extracts to the skin. Preferably a nutritionalsignal in the extract reaches and is effective as a nutrient to the skincells. Preferably the composition is in a water based gel comprisinghyaluronic acid and/or chitosan. In another preferred embodiment, theextract is a spray acting as a liquid band-aid or fluid that dries onthe skin. In further embodiments, the liquid contains gel-formingcomponents such as collagen and chitosan. In further preferredembodiments, the composition is a component of a film on a support orcream.

The present invention also provides for use of the foregoingcompositions for the treatment of skin, for removing wrinkles, forrejuvenation of skin, for wound healing, for improving the appearance ofskin, the prevent damage to skin, to prevent deterioration of skin, orto provide nutrients to skin and any other use described herein.

The present invention further provides methods for preparing acomposition for topical application to the skin comprising: providingdifferentiable cells or preparing an extract or fraction ofdifferentiable cells or eggs; and formulating said differentiable cellsor said extract with an agent for topical administration to the skin toprovide a cream, gel, spray, emulsion, solid, plastic or matrix,ointment, powder or lotion suitable for topical administration. Infurther embodiments, the present invention provides compositions made bythe foregoing methods.

In some embodiments, the present invention provides methods comprising:contacting the skin of said subject with a cellular extract in an amounteffective to cause one or more effects selected from the groupconsisting of reduction of fine lines in the skin, normalization of skincolor, increasing skin water content and hydration, decreasing ornormalizing the amount of sebum in the skin, decreasing production ofmelanin, increasing collagen protein production, increasing collagengene expression, increasing adult stem cell proliferation, increasingcellular metabolism of carbohydrates, increasing cellular metabolism oflipids, prevention of apoptosis, increasing angiogenesis, upregulationthe cell cycle of cells, increasing angiogenesis, increasing the haircycle, increasing follicular development, and increasing cellproliferation. In some embodiments, the effect is upregulation of a genelisted in Table 11 or regulation or upregulation of a pathway or effectlisted in Table 11. In some embodiments, the present invention providesfor the use of a cellular extract for one or more of reduction of finelines in the skin, normalization of skin color, increasing skin watercontent and hydration, decreasing or normalizing the amount of sebum inthe skin, decreasing production of melanin, increasing collagen proteinproduction, increasing collagen gene expression, increasing adult stemcell proliferation, increasing cellular metabolism of carbohydrates,increasing cellular metabolism of lipids, prevention of apoptosis,increasing angiogenesis, upregulation the cell cycle of cells,increasing angiogenesis, increasing the hair cycle, increasingfollicular development, and increasing cell proliferation. In someembodiments, the effect is upregulation of a gene listed in Table 11 orregulation or upregulation of a pathway or effect listed in Table 11. Insome embodiments, the effect is on one or more types of cell-typesassociated with the skin. In some embodiments, the cell type associatedwith the skin is selected from the group consisting of a keratinocyte,fibroblast, melanocyte, and adipocyte.

In some embodiments, normalization of skin color comprises a reductionof skin erythema index. In some embodiments, the reduction in the skinmelanin index or erythema index is measured by a skin analysis system,such as the Mexameter (MX18, Courage+Khazaka, Germany), using theprotocol provided with the system. In some embodiments, the cellularextract comprises about 100 to 380 mg/ml protein in an aqueous solution;about 0.1 to 10 mg/ml RNA; about 0.1 to 5 mg/ml DNA and 0.1-10% lipidsw/w; wherein said composition has an osmolarity of from about 330 to 440mOsm, a pH of from about 5.0 to 7.7, and density of from about 0.8 to1.4 g/ml. In some embodiments, the cellular extract is selected from thegroup consisting of an extract of an activated fish egg cellular extractand an unactivated fish egg cellular extract. In some embodiments, thefish egg cellular extract is from a fertilized egg. In some embodiments,the cellular extract is provided in a cream, gel, emulsion, ointment,spray, powder or lotion. In some embodiments, the cellular extract is acytoplasmic extract.

In some embodiments, the present invention provides a compositioncomprising a cellular extract and an agent selected from the groupconsisting of Vitamin C and iron. In some embodiments, the cellularextract comprises about 100 to 380 mg/ml protein in an aqueous solution;about 0.1 to 10 mg/ml RNA; about 0.1 to 5 mg/ml DNA and 0.1-10 lipidsw/w; wherein said composition has an osmolarity of from about 330 to 440mOsm, a pH of from about 5.0 to 7.7, and density of from about 0.8 to1.4 g/ml. In some embodiments, the cellular extract is selected from thegroup consisting of an extract of an activated fish egg cellular extractand an unactivated fish egg cellular extract. In some embodiments, thefish egg cellular extract is from a fertilized egg. In some embodiments,the cellular extract is provided in a cream, gel, emulsion, ointment,spray, powder or lotion. In some embodiments, the cellular extract is acytoplasmic extract.

In some embodiments, the present invention provides processescomprising: treating fish eggs to reduce bacterial load; homogenizingsaid fish eggs by application of pressure to produce a fish egghomogenate; and separating an active fraction from said fish egghomogenate by centrifugation, wherein said active fraction comprisesabout 100 to 380 mg/ml protein in an aqueous solution; about 0.1 to 10mg/ml RNA; about 0.1 to 5 mg/ml DNA and 0.1-10% lipids w/w. In someembodiments, the pressure is hydraulic pressure. In some embodiments,the pressure is about 5 to about 30 tons. In some embodiments, thecentrifugation is continuous.

In some embodiments, the present invention provides a process forproducing an active fish egg fraction comprising: milling the fish eggsbetween two surfaces, at least one of which is a milling surface,wherein the surfaces have a space there between so that said fish eggsare crushed when passed between the surfaces to provide a fish egghomogenate; and separating an active fraction from said fish egghomogenate, wherein said active fraction comprises about 100 to 380mg/ml protein in an aqueous solution; about 0.1 to 10 mg/ml RNA; about0.1 to 5 mg/ml DNA and 0.1-10% lipids w/w. In some embodiments, themilling surface comprising cutting elements. In some embodiments, thecutting elements comprise knurls. In some embodiments, the surfaces arecylindrical and rotate. In some embodiments, the surfaces are separatedby about 0.1 to 2.0 mm. In some embodiments, the separating comprisingcentrifugal separation.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of generations over time.

FIG. 2 is a graph of minutes/generation v. time.

FIG. 3 is a growth curve graph.

FIG. 4 provides a graph demonstrating the effect of LEX on proliferationof fibroblasts in vitro. Diamond—control, square—LEX6, triangle—LEX15.

FIG. 5 provides a graph of the fold induction of trout roe, unfertilizedsalmon egg (salmon roe) and fertilized salmon egg (eyeroe) extracts.

FIG. 6: Collagen secretion from human fibroblasts is increased by 500%in one week in vitro. *** p<0.001.

FIG. 7: As the body ages, fewer new cells are produced. LEX can reversethis effect by increasing fibroblast cell number in vitro. * p<0.05.

FIG. 8: 5% extract in a serum base gives significant decrease in surfaceroughness and fine lines over control at day 7, 14 and 28. 1% extracthas a significant effect over control at day 7. (Average values withingroups shown).

FIG. 9: 5% extract (LEX) is significantly better at reducing melanin inthe skin to control.

FIG. 10: 5% extract (LEX) is significantly better at reducing redness inthe skin to control.

FIG. 11: 5% extract (LEX) is significantly better at improving lucidity(L-value) of the skin to control.

FIG. 12: Water content of the skin is increased significantly by 5%extract compared to control.

FIG. 13: 5% extract decreases sebum content in the skin compared tocontrol.

FIG. 14: Fold change in cells per flask from control to LEX stimulatedcells and fold change melanin per cell from control to LEX stimulatedcells.

FIG. 15: Comparison of collagen production by retinoic acid and LEXtreated cells.

FIG. 16: Comparison of collagen production by retinoic acid and LEXtreated cells.

FIG. 17: The absorbance values of collagen present in cell medium fromcells stimulated with LEX compared to control cells. The figure shows a3.42 fold induction of collagen in the medium of LEX stimulated cells(0,167) compared to control cells (0,049), The data are presented as anaverage (n=3)±SEM (standard error of the mean).

FIG. 18: Collagen absorbance measured for hsF cells both in monoculture(red) and co-culture with HEM cells (blue). Collagen was measured bothafter LEX stimulation for eight days and for non-stimulated controlcells.

FIG. 19: Melanin levels per melanocyte cell after eight days ofhsf-co-culture with or without LEX stimulation.

FIG. 20: Absorbance measurements on XTT proliferation assay. ADSCstimulated with 0.1, 0.25, 0.5 and 1% LEX+ADSC un-stimulated control.Samples stimulated for 3 days and measurements done over 2 to 12 hours.FIG. 1 using baseline 650 nm.

FIG. 21: Absorbance measurements on XTT proliferation assay. ADSCstimulated with 0.1, 0.25, 0.5 and 1% LEX and control using 1% FBS.Samples stimulated for 3 days and measurements done over 8 hours, usingbaseline 650 nm. * P<0.05 and ** P<0.05 for 0.5% and 1.0%

FIG. 22: Absorbance measurements on XTT proliferation assay. ADSCstimulated with 0.1, 0.25, 0.5 and 1% LEX+ADSC un-stimulated controlusing 10% FBS. Samples stimulated for 3 days and measurements done over8 hours, using baseline 650 nm. * P<0.05 and ** P<0.05 for 0.1%, 0.25%and 1.0%

DEFINITIONS

“Anti-infective agents” include, but are not limited tobenzylpenicillin, penicillin, enicillin G, 6-phenyl acetyl penicillin,penicillin V, micronomicin, clavulanate, oxacillin, dequalinium,cloxacillin, sulbenicillin, ampicillin, cilleral, and principen andcombinations thereof.

“Anti-inflammatory” means a substance that reduces inflammation. Manyanalgesics remedy pain by reducing inflammation. Manysteroids—specifically glucocorticoids—reduce inflammation by binding tocortisol receptors. Non-steroidal anti-inflammatory drugs (NSAIDs)alleviate pain by counteracting the cyclooxygenase (COX) enzyme. On itsown COX enzyme synthesizes prostaglandins, creating inflammation. Manyherbs have anti-inflammatory qualities, including but not limited tohyssop and willow bark (the latter of which contains salicylic acid, theactive ingredient in aspirin), as well as birch, licorice, wild yam andginseng.

“Antioxidants” means any of a variety of substances that prevent or slowthe breakdown of another substance by oxygen. Synthetic and naturalantioxidants are used to slow the deterioration of gasoline and rubber,and such antioxidants as vitamin C (ascorbic acid), butylatedhydroxytoluene (BHT), and butylated hydroxyanisole (BHA) are typicallyadded to foods to prevent them from becoming rancid or from discoloring.Nutrients such as beta-carotene (a vitamin A precursor), vitamin C,vitamin E, and selenium have been found to act as antioxidants. They actby scavenging free radicals, molecules with one or more unpairedelectrons, which rapidly react with other molecules, starting chainreactions in a process called oxidation. Free radicals are a normalproduct of metabolism; the body produces its own antioxidants (e.g., theenzyme superoxide dismutase) to keep them in balance. However, stress,aging, and environmental sources such as polluted air and cigarettesmoke can add to the number of free radicals in the body, creating animbalance. The highly reactive free radicals can damage healthy DNA andhave been linked to changes that accompany aging (such as age-relatedmacular degeneration, a leading cause of blindness in older people) andwith disease processes that lead to cancer, heart disease, and stroke.

An “antiseptic” is a substance that kills or prevents the growth andreproduction of various microorganisms, including bacteria, fungi,protozoa, and viruses on the external surfaces of the body. Theobjective of antiseptics is to reduce the possibility of sepsis,infection or putrefaction by germs. Antibacterials have the sameobjective but only act against bacteria. Antibiotics perform a similarfunction, preventing the growth or reproduction of bacteria within thebody. Antiseptics include, but are not limited to, alcohol, iodine,hydrogen peroxide, and boric acid. There is great variation in theability of antiseptics to destroy microorganisms and in their effect onliving tissue. For example, mercuric chloride is a powerful antiseptic,but it irritates delicate tissue. In contrast, silver nitrate killsfewer germs but can be used on the delicate tissues of the eyes andthroat. There is also a great difference in the time required fordifferent antiseptics to work. Iodine, one of the fastest-workingantiseptics, kills bacteria within 30 sec. Other antiseptics haveslower, more residual action. Since so much variability exists, systemshave been devised for measuring the action of an antiseptic againstcertain standards. The bacteriostatic action of an antiseptic comparedto that of phenol (under the same conditions and against the samemicroorganism) is known as its phenol coefficient.

“Chitosan” is a beta-1,4-linked glucosamine polymer which, unlikechitin, contains few, if any, N-acetyl residues. It may be obtained fromchitin, a polysaccharide found in the exoskeletons of crustaceans suchas shrimp, lobster, and crabs. The shells may be ground into a pulverouspowder. This powder is then deacetylated which allows the chitosan toabsorb lipids.

“Collagen” means any of a variety of substances that contains the alphachains of the collagen polypeptide with a sequence that generallyfollows the pattern Gly-X-Y, where Gly for glycine, X for proline, and Yfor proline or hydroxyproline. Collagen proteins also containsignificant amounts of glycine and proline. Hydroxyproline andhydroxylysine are not inserted directly by ribosomes. They arederivatised from proline and lysine in enzymatic processes ofpost-translational modification, for which vitamin C is required. Thisis related to why vitamin C deficiencies can cause scurvy, a diseasethat leads to loss of teeth and easy bruising caused by a reduction instrength of connective tissue due to, a lack of collagen, or defectivecollagen. Cells called fibroblasts form the various fibers in connectivetissue in the body including collagen. The white collagen that makes upthe matrix of most connective tissue in mammals consists of inter-wovenfibres of the protein collagen. The collagen fibers consist of globularunits of the collagen sub-unit, tropocollagen. Tropocollagen sub-unitsspontaneously arrange themselves under physiological conditions intostaggered array structures stabilized by numerous hydrogen and covalentbonds. Tropocollagen sub-units are left-handed triple helices where eachstrand is, further, a right-handed helix itself. Thus, tropocollagen maybe considered to be a coiled coil.

Although collagen is responsible for skin elasticity, and itsdegradation leads to wrinkles that accompany aging, it occurs in manyother places throughout the body, and in different forms known as types:Type I collagen—This is the most abundant collagen of the human bodypresent in scar tissue, the end product when tissue heals by repair;Type II collagen—Auricular cartilage Type III collagen—This is thecollagen of granulation tissue, and is produced quickly by youngfibroblasts before the tougher type I collagen is synthesized; Type IVcollagen—Basal lamina; Type V collagen—most interstitial tissue, assoc.with type I; Type VI collagen—most interstitial tissue, assoc. with typeI; Type VII collagen—epithelia; Type VIII collagen—some endothelialcells; Type IX collagen—cartilage, assoc. with type II; Type Xcollagen—hypertrophic and mineralizing cartilage; Type XIcollagen—cartilage; Type XII collagen—interacts with types I and III.

With in the context of certain embodiments, “collagen modulatingsubstances” means a variety of substances capable of facilitating theformation or breaking down of units or of any type of collagen.

A “gel” is a semisolid material formed from a colloidal solution. Byweight, gels are mostly liquid, yet they behave like solids. An exampleis gelatin.

“Keratin” is any of a variety of fibrous protein molecules that serve asstructural units for various living tissues. The keratins are the majorprotein components of hair, wool, nails, horn, hoofs, and the quills offeathers. These proteins generally contain large quantities of thesulfur-containing amino acids, particularly cysteine. The helicalkeratin molecules twist around each other to form elongated strandscalled intermediate filaments. The formation of a disulfide bridgebetween the sulfur atoms on two cysteines on separate polypeptide chainsof keratin allows for the cross-linkage of these chains and results in afairly rigid aggregate.

“Filaggrin” is any of a variety of filament-associated proteins thatinteract with keratin intermediate filaments of terminallydifferentiating mammalian epidermis via disulphide bond formation.

“Immunomodulator” means any of a variety of substance that influencesthe immune system. Examples include, but are not limited to, cytokines,Interleukin-2, immunostimulants, and immunosuppressors.

The term “natural product” means any of a variety of organic chemicalmoieties whose molecular arrangement is derived from enzymatictransformations in a living organism excluding amino acids, proteins,polypeptides, nucleic acids and sequences, and saturated fatty acids.Examples include, but are not limited to lipids (i.e., that are notsaturated fatty acids), carbohydrates/saccharides and polysaccharides,the steroids and their derivatives, the terpenes and their derivatives,vitamins, carotenoids, and natural medicines such as taxol, etc. Theterm “synthetic natural product” is a natural product not obtained fromits natural source.

The term “gene” as used herein, refers to a DNA sequence that comprisescontrol and coding sequences necessary for the production of apolypeptide or protein precursor. The polypeptide can be encoded by afull length coding sequence or by any portion of the coding sequence, aslong as the desired protein activity is retained.

“Nucleoside,” as used herein, refers to a compound consisting of apurine [guanine (G) or adenine (A)] or pyrimidine [thymine (T), uridine(U), or cytidine (C)] base covalently linked to a pentose, whereas“nucleotide” refers to a nucleoside phosphorylated at one of its pentosehydroxyl groups.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin that may be single- ordouble-stranded, and represent the sense or antisense strand.

An “amino acid sequence” as used herein refers to a peptide or proteinsequence.

A “peptide nucleic acid” as used herein refers to an oligomeric moleculein which nucleosides are joined by peptide, rather than phosphodiester,linkages. These small molecules, also designated anti-gene agents, stoptranscript elongation by binding to their complementary (template)strand of nucleic acid (Nielsen et al. (1993) Anticancer Drug Des.,8:53-63).

“Peptides”, herein defined as polymers formed from the linking, in adefined order, of α-amino acids; including but not limited to milkpeptides, ribosomal peptides, nonribosomal peptides, peptones andpeptide fragments. Peptides are believed to have a good effect on skinand wrinkles. Because peptides are so small, it is thought that they maymore easily penetrate the skin and yield their effects.

A “variant” in regard to amino acid sequences is used to indicate anamino acid sequence that differs by one or more amino acids fromanother, usually related amino acid. The variant may have “conservative”changes, wherein a substituted amino acid has similar structural orchemical properties (e.g., replacement of leucine with isoleucine). Morerarely, a variant may have “non-conservative” changes, e.g., replacementof a glycine with a tryptophan. Similar minor variations may alsoinclude amino acid deletions or insertions (i.e., additions), or both.Guidance in determining which and how many amino acid residues may besubstituted, inserted or deleted without abolishing biological orimmunological activity may be found using computer programs well knownin the art, for example, DNAStar software.

As used herein the term “portion” in reference to an amino acid sequenceor a protein (as in “a portion of an amino acid sequence”) refers tofragments of that protein. The fragments may range in size from fouramino acid residues to the entire amino acid sequence minus one aminoacid.

As used herein, the term “purified” refers to molecules, including butnot limited to nucleic, ribonucleic, lipid or amino acid sequences,which are removed from their natural environment, isolated or separated.An “isolated nucleic acid sequence” is therefore a purified nucleic acidsequence. “Substantially purified” molecules are at least 60% free,preferably at least 75% free, and more preferably at least 90% free fromother components with which they are naturally associated.

“Cancer” means any of various cellular diseases with malignant neoplasmscharacterized by the proliferation of anaplastic cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Most cancers arenamed for the type of cell or organ in which they start.

“Cell” means the smallest structural unit of living matter capable offunctioning autonomously, consisting of one or more nuclei, cytoplasm,and various organelles, all surrounded by a semipermeable membrane.Cells include all somatic cells obtained or derived from a living ordeceased animal body at any stage of development as well as germ cells,including sperm and eggs (animal reproductive body consisting of an ovumor embryo together with nutritive and protective envelopes). Includedare both general categories of cells: prokaryotes and eukaryotes. Thecells contemplated for use in this invention include all types of cellsfrom all organisms in all kingdoms: plans, animals, protists, fungi,archaebacteria and eubacteria. Stem cells are cells capable, bysuccessive divisions, of producing specialized cells on many differentlevels. For example, hematopoietic stem cells produce both red bloodcells and white blood cells. From conception until death, humans containstem cells, but in adults their power to differentiate is reduced.

As used herein, the term “differentiation” related to cells means theprocess by which cells becomes structurally and functionallyspecialized, which is a progressive restriction of the developmentalpotential and increasing specialization of function which takes placeduring the development of the embryo and leads to the formation ofspecialized cells, tissues, and organs.

The term “dedifferentiation” related to cells means the reverse processof differentiation, where cells become less structurally andfunctionally specialized, which increases the developmental potential ofthe cell.

“Differentiable” means the ability of a cell to differentiate into adesired cell type. As used herein, the term “differentiates” meansspecialization (differentiation) or return to a more primitive celltype; dedifferentiation).

An “extract” as used in the context of “cell extract” and “egg extract”in this invention means a preparation of any type of cell as definedabove obtained by chemical or mechanical action, as by pressure,distillation, evaporation etc. Extracts can include all or any singlecomponent or combination of components of the cells, includingconcentrated preparations of the active components. Such components ofthe extracts include but are not limited to RNA, DNA, lipids, all aminoacid base structures including peptides and proteins, carbohydrates orcombinations thereof. Extracts contemplated by this invention includebut are not limited to extracts of fish eggs, urchin eggs, frog eggs,adult stem cells, plant seeds and plant stem cells.

“Growth media” are compositions used to grow microorganisms or cells inculture. There are different sorts of media for growing different sortsof cells. The biggest difference in growth media are between those usedfor growing cells in culture (cell culture uses specific cell typesderived from plants or animals) and those used for growingmicroorganisms (usually bacteria or yeast). These differences arise dueto the fact that cells derived from whole organisms and grown in cultureare often incapable of growth without the provision of certainrequirements, such as hormones or growth factors which usually occur invivo. In the case of animal cells these requirements are often providedby the addition of blood serum to the medium. These media are often redor pink due to the inclusion of pH indicators. Growth media forembryonic stem cells preferably contains minimal essential medium, i.e.,Eagle's: amino acids, salts (Ferric nitrate nonahydrate, Potassiumchloride, Magnesium sulfate, Sodium chloride, Sodium dihydrogenphosphate), vitamins, (Ascorbic acid, Folic acid, Nicotinamide,Riboflavin, B-12) or Dulbecco's: additionally iron, glucose;non-essential amino acids, sodium pyruvate, β-mercaptoethanol,L-glutamine, fetal bovine serum and Leukemia Inhibitory Factor (LIF). Inthe case of microorganisms, there are no such limitations as they areoften single cell organisms. One other major difference is that animalcells in culture are often grown on a flat surface to which they attach,and the medium is provided in a liquid form, which covers the cells.Bacteria such as Escherichia coli (E. coli, the most commonly usedmicrobe in laboratories) may be grown on solid media or in liquid media,liquid nutrient medium is commonly called nutrient broth. The preferredgrowth media for microorganisms are nutrient broth or Luria-Bertanimedium (L-B medium). Bacteria grown in liquid cultures often formcolloidal suspensions. When agar (a substance which sets into a gel) isadded to a liquid medium it can be poured into Petri dishes where itwill solidify (these are called agar plates) and provide a solid mediumon which microbes may be cultured.

Within the context of certain embodiments, “to glue to skin” means tostick or fasten to with or as if with any of various adhesives, such as,glue, paste or mucilage.

A “lipid” means any of a group of organic compounds, including the fats,oils, waxes, sterols, and triglycerides that are insoluble in water butsoluble in nonpolar organic solvents, and are oily to the touch. Majorclasses of lipids include the fatty acids, the glycerol-derived lipids(including the fats and oils and the phospholipids), thesphingosine-derived lipids (including the ceramides, cerebrosides,gangliosides, and sphingomyelins), the steroids and their derivatives,the terpenes and their derivatives, certain aromatic compounds, andlong-chain alcohols and waxes. In living organisms lipids serve as thebasis of cell membranes and as a form of fuel storage. Often lipids arefound conjugated with proteins or carbohydrates, and the resultingsubstances are known as lipoproteins and lipopolysaccharides. Thefat-soluble vitamins can be classified as lipids. Liposomes arespherical vesicles formed by mixing lipids with water or watersolutions. They have found applications in the oral administration ofsome drugs (e.g., insulin and some cancer drugs), since they retaintheir integrity until they are broken down by the lipases in the stomachand small intestine.

Within the context of certain embodiment, a “nutrient gel layer” a gelcomprising substances typically contained in a growth medium.

Within the context of certain embodiments, “specialized cell” of asubject means that the cell has characteristic immuno-identificativemarkers, such that differentiation of these cells and exposure totissues of the subjects can be done under conditions such that immunesystem does not create antibodies to the differentiated cells. Forexample, when red blood cells carrying one or both A or B antigens areexposed to the corresponding antibodies, they agglutinate; that is,clump together. People usually have antibodies against those red cellantigens that they lack. Thus, specialized red blood cells of thesubject would be those of the proper blood type. The cause of transplantrejection is recognition of foreign MHC antigens by T cells andactivation of those T cells to become effector cytotoxic or helper Tcells. T cell activation occurs in the case of vascularized grafts ofnucleated cells expressing MHC Matching MHC Class I (especially HLA-B)and Class II HLA-DR alleles is more important for successfultransplantation than matching other MHC antigens; and matching MHC ismore important than matching minor histocompatibility antigens. Thus,specialized MHC presenting cells of the subject would be thosepresenting matching MHC alleles.

The term “manage” when used in connection with a disease or conditionmeans to provide beneficial effects to a subject being administered witha prophylactic or therapeutic agent, which does not result in a cure ofthe disease. In certain embodiments, a subject is administered with oneor more prophylactic or therapeutic agents to manage a disease so as toprevent the progression or worsening of the disease.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present invention be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, the present invention also contemplates treatmentthat merely reduces symptoms, and/or delays disease progression.

Within the context of certain embodiments, a “waterproof layer” means amaterial or fabric that is substantially impervious to water or a layerof a sealing agent to intended to prevent substantial penetration bywater.

As used herein, the term “transport vehicle” includes substances capableof aiding penetration of intact skin or skin cells or other somaticcells. The term “transport vehicle” is used synonymously with the term“permeabilizing agents”. Such transport vehicles include, but are notlimited to: phospholipids, palmitylmyristyrates, DMSO, polymer orchitosan suspensions or matrix, liposomes, Trojan peptides, chariotpeptides, small elastic vesicles, microspheres (functionalized vectorsmade from naturally derived materials such as collagen,glycosaminoglycans, chondroitin sulfate, chitosan or polysaccharides),nanoparticles (carries lipophilic substances and enhance bioavailabilityof the encapsulated material into skin), preloaded spherical beads andsponges, uni- and/or multilamellar vesicles (stabilize contents ofextracts in cream base and help transport into skin), retinol molecularfilm fluid (thin uniform monolayer film that facilitates the transfer ofactives through the stratum corneum), poly acrylo nitrile (polymerscomprising a controlled release system that synchronizes the release ofan active ingredient along with a fragrance as a sensory marker whichconveys the efficacy of the product), beta-glucan (oat fiber which aidsin penetration of the skin, (Redmond, Int. Journ. Cosmetic science2005), propylene glycol (as drug carrier, work best with a mineral oilbased cream/lotion etc), butylene glycol, polyethylene glycol, oliveoil, dimethyl isosorbide, dimethylformamide, methyl salicylate (theseall enhance absorption through skin), long chain oleic acids (disruptsthe bilayer within the stratum corneum, vital for permeation ofcompositions in propylene glycol-based formulations), substances capableof adjusting pH, hydration and local metabolism in skin. Agentsmodifying these factors include a vehicle containing an activehydrophobic agent, de-ionization of active ingredients, increasedhydration of the skin (water content of carrier solution/cream/medium),lactic acid (alters the pH).

As used herein, the term “NANOG” refers to a homeobox gene. NANOG isthought to be required for stem cells to multiply without limit whileremaining able to make many different types of cells. The gene is apotential master gene that helps make embryonic stem cells grow in thelaboratory, making stem cells immortal.

As used herein, the term “OCT4” refers to a gene that is not active insomatic cells, including adult stem cells, but is expressed in embryonicstem and germ cells. OCT4 is essential to maintain pluripotency ofembryonic stem cells.

As used herein, the term “SOX2” refers to the sex determining region Y(SRY) box 2 protein coding gene. This intronless gene encodes a memberof the SRY-related HMG-box (SOX) family of transcription factorsinvolved in the regulation of embryonic development and in thedetermination of cell fate.

As used herein, the term “GAPDH” refers to the housekeeping geneglyceraldehydes-3-phosphate dehydrogenase. This gene is involved inbasic functions needed for cell maintenance. Housekeeping genes areconstitutively expressed.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to improving health and damage of cells andtissues preferably skin, and more preferably restoring aged or damagedskin to a youthful and healthy appearance. In some embodiments, theinvention relates to compositions of cells, cell or egg extracts, andextract components which can induce differentiation, including but notlimited to purified or synthetic nucleic acid sequences, polypeptides,or natural products contained in said extracts. In some embodiments, thecells are differentiable cells, preferably stem cells or eggs. In somepreferred embodiments, the extracts are aqueous extracts. In someembodiments, the extracts are from a non-avian source. In someembodiments, compositions are used in a method that comprisesapplication of compositions to skin and/or wounds after removal theouter surface layers. In some embodiments, the invention related to amethod of dedifferentiation of cells and/or dedifferentiation followedby redifferentiation. In some embodiments, the invention relates tomanaging, preventing, and treating skin diseases.

Application of the composition to the desired surface may beprophylactic, so that the composition is applied to the skin or othersurface before exposure to an agent occurs. Application of thecomposition may be curative, for example, to further protect acompromised skin surface or to provide a protectant surface duringnatural or mediated healing of an exposed skin surface. Application ofthe composition may be protective, for example, to protect a skinsurface should exposure to the agent occur.

The present invention relates to the use of extracts or components ofdifferentiable cells for topical application to surfaces of the body.Accordingly, the present invention provides methods and compositions forcosmetic and therapeutic uses. The present invention is not limited tothe use of extracts or components of any particular type ofdifferentiatable cell. Indeed, the use of variety of types of cells anddifferentiable cells from any organism is contemplated, including, butnot limited to, mammalian embryonic stem cells, mammalian adult stemcells, cord blood cells, fish, shrimp or sea urchin eggs and embryos,and amphibian eggs and embryos.

In some embodiments, the invention relates to dedifferentiating existingepithelial/epidermal cells to a primordial state, wherein the cells havestem-cell capacities and can reform the correct and needed cells for theregeneration of the whole layer of skin (epidermis, dermis andsubdermis). Although many differentiated cells are typically committedto their fate, dedifferentiation events can take place. Urodeleamphibians and teleost fish can replace lost anatomical parts by aprocess of migration, dedifferentiation, proliferation andredifferentiation of epithelial cells in the wounded area. Functionalreprogramming of differentiated cell nuclei has also been illustrated bythe derivation of pluripotent embryonic stem cells (ESCs), and by thelive birth of cloned animals after nuclear transplantation intounfertilized eggs.

The term plasticity, as used in this herein, means that a cell from onetissue can generate the differentiated cell types of another tissue.Xenopus eggs can reprogram mammalian somatic nuclei to express the POUfamily member homeodomain transcription factor gene Oct4 by a processrequiring DNA demethylation. DNA demethylation also occurs after fusionof mouse thymocytes with embryonic germ cells (EGCs) but interestingly,only EG cells are capable of demethylating imprinted genes. Fusion ofneuronal progenitor cells or bone marrow derived cells with ESCs resultsin hybrids which express markers of pluripotency. Similar results areobtained from fusing human fibroblasts with ESCs. Fusion of embryonalcarcinoma cells (ESCs) with T-lymphoma cells also promotes the formationof colonies expressing pluripotent cell transcripts from the lymphomagenome. Components of pluripotent EG, ES or EC cells can elicitreprogramming events in a somatic genome.

Somatic nuclear function can be altered using nuclear and cytoplasmicextracts because extracts provide the necessary regulatory components.Extracts of regenerating newt limbs promote cell cycle reentry anddownregulation of myogenic markers in differentiated myotubes.Teratocarcinomas are a particular type of germ cell tumors which containundifferentiated stem cells and differentiated derivatives that caninclude endoderm, mesoderm and ectoderm germ layers. Undifferentiatedcarcinoma cells can be cultured to give rise to lines of ECCs. ECCs formmalignant teratocarcinomas when transplanted into ectopic sites;however, some ECC lines can also contribute to tissues of the developingfetus when introduced into a blastocyst.

Undifferentiated human teratocarcinoma NCCIT cells can be establishedfrom a mediastinal mixed germ cell tumor. NCCIT is at a stageintermediate between a seminoma (a precursor of germ cell tumors) and anembryonal carcinoma. NCCIT is a developmentally pluripotent cell linethat can differentiate into derivatives of all three embryonic germlayers and extraembryonic cell lineages an extract of undifferentiatedsomatic cells can elicit dedifferentiation in a somatic cell line. SeeTaranger et al., “Induction of Dedifferentiation, Genome-wideTranscriptional Programming, and Epigenetic Reprogramming by Extracts ofCarcinoma and Embryonic Stem Cells” Mol Biol Cell. (2005).

Stem cells can establish in damaged tissue. See Menard et al.,“Transplantation of cardiac-committed mouse embryonic stem cells toinfarcted sheep myocardium: a preclinical study” Lancet,366(9490):1005-12 (2005); Goldman “Stem and progenitor cell-basedtherapy of the human central nervous system” Nat Biotechnol.23(7):862-71 (2005); Leri et al., “Repair of the damaged heart” KidneyInt. 68(5):1962 (2005); Levy et al., “Embryonic and adult stem cells asa source for cell therapy in Parkinson's disease” J Mol Neurosci.24(3):353-86 (2004); Jack et al., “Processed lipoaspirate cells fortissue engineering of the lower urinary tract: implications for thetreatment of stress urinary incontinence and bladder reconstruction” JUrol. 174(5):2041-5 (2005); Kitmaura et al., Establishment of renalstem/progenitor-like cell line from S3 segment of proximal tubules inadult rat kidney Kidney Int. 68(5):1966 (2005).

In some embodiments, the invention relates to extracts that are capableof stimulating the immune system to aid in healing. For example, theextracts may contain fibrogen and heat shock proteins. These endogenouscellular components are alarm signals typically expressed in distressedor injured cells. They bind Toll-like receptors (TLRs) in antigenpresenting cells (APCs) and put the immune system on alert of a damagedarea. See Matzinger “The Danger Model: A Renewed Sense of Self” Science296:301-305 (2002).

In some embodiments, the invention relates to stimulating existing stemcells in skin, such as stem cells found in and around hair follicles toduplicate and/or differentiate into epithelial cells or neurons. Nestin,a marker for neural progenitor cells, is expressed in cells of thehair-follicle bulge and behave as stem cells, differentiating to formmuch of the hair follicle during each hair growth cycle. The hairfollicle is dynamic, cycling between growth (anagen), regression(catagen), and resting (telogen) phases throughout life. Stem cellslocated in the hair-follicle bulge area give rise to the folliclestructures during each anagen phase. Bulge hair-follicle stem cells cangenerate all epithelial cell types within the intact follicle and hairduring normal hair-follicle cycling. The bulge hair-follicle stem cellsdifferentiate into hair-follicle matrix cells, sebaceous-gland basalcells, and epidermis. In response to wounding, some stem cells exit thebulge, migrate, and proliferate to repopulate the infundibulum andepidermis. Multipotent adult stem cells from the skin dermis, termedskin-derived precursors (SKPs), can proliferate and differentiate toproduce neurons, glia, smooth muscle cells, and adipocytes. Pluripotentneural crest stem cells are present in the dermal papillae of adultmammalian hair follicles. See Amoh et al., “Multipotent nestin-positive,keratin-negative hair-follicle bulge stem cells can form neurons” ProcNatl Acad Sci USA. 12; 102(15):5530-4 (2005).

The bone marrow contains three stem cell populations—hematopoietic stemcells, stromal cells, and endothelial progenitor cells. Bone marrow stemcells, the hematopoietic stem cells (HSCs), are responsible for formingall of the types of blood cells in the body. The bone marrow-derivedcells are sometimes sorted—using a panel of surface markers—intopopulations of hematopoietic stem cells or bone marrow stromal cells.The HSCs may be highly purified or partially purified, depending on theconditions used. Another way to separate population of bone marrow cellsis by fractionation to yield cells that adhere to a growth substrate(stromal cells) or do not adhere (hematopoietic cells). The mesenchymalstem cells of the bone marrow also give rise to these tissues, andconstitute the same population of cells as the bone marrow stromalcells. Progenitor cells that differentiate into endothelial cells, atype of cell that lines the blood vessels, can be isolated fromcirculating blood. Pericytes are related to bone marrow stromal cells.

Combinations of surface markers are used to identify, isolate, andpurify HSCs derived from bone marrow and blood. Undifferentiated HSCsand hematopoietic progenitor cells express c-kit, CD34, and H-2K. Thesecells usually lack the lineage marker Lin, or express it at very lowlevels (Lin−/low). BM stromal cells have several features thatdistinguish them from HSCs. The two cell types are separable in vitro.When bone marrow is dissociated, the mixture of cells it contains isplated at low density, the stromal cells adhere to the surface of theculture dish, and the HSCs do not. Given specific in vitro conditions,BM stromal cells form colonies from a single cell called the colonyforming unit-F (CFU-F). These colonies may then differentiate asadipocytes or myelo supportive stroma, a clonal assay that indicates thestem cell-like nature of stromal cells. Unlike HSCs, which do not dividein vitro (or proliferate only to a limited extent), BM stromal cells canproliferate for up to 35 population doublings in vitro. Endothelial stemcells are CD34+(a marker for HSCs), and they express the transcriptionfactor GATA-2 see Kocher, et al., “Neovascularization of ischemicmyocardium by human bone-marrow-derived angioblasts preventscardiomyocyte apoptosis, reduces remodeling and improves cardiacfunction” Nat. Med. 7, 430-436 (2001).

The present invention contemplates the use of any type of cell includingstem cells from any multicellular organism in any kingdom of species,both eukaryotes including animals, plants, protists, fungi, andprokaryotes including the kingdoms archaebacteria and eubacteria.Multicellular organisms contain totipotent, mulitpotent, pluripotent andunipotent stem cells capable of dividing and replenishing tissues andcells which compose the organism. Stem cells are well documented inmammalian animals, but are present in all animals, e.g. insects. Adultfruit flies have the same stem cells controlling cell regulation intheir gut as humans do. Vertebrate and invertebrate digestive systemsshow extensive similarities in their development, cellular makeup andgenetic control. The Drosophila midgut is typical: enterocytes make upthe majority of the intestinal epithelial monolayer, but areinterspersed with hormone-producing enteroendocrine cells. Human (andmouse) intestinal cells are continuously replenished by stem cells, themisregulation of which may underlie some common digestive diseases andcancer. In contrast, stem cells have not been described in theintestines of flies, and Drosophila intestinal cells have been thoughtto be relatively stable. By lineage labelling it has been shown thatadult Drosophila posterior midgut cells are continuously replenished bya distinctive population of intestinal stem cells (ISCs). (BenjaminOhlstein and Allan Spradling, The adult Drosophila posterior midgut ismaintained by pluripotent stem cells, Nature, online Dec. 7, 2005).

In addition to animal stem cells, plants also contain stem cells. Stemcells in plant shoot and root meristems are maintained throughout thelife of the plant and produce somatic daughter cells that make up thebody of the plant. Plant stem cells can also be derived from somaticcells in vivo and in vitro. (Plants stem cells: divergent pathways andcommon themes in shoots and roots. Byrne M E, Kidner C A, Martienssen RA. Curr Opin Genet Dev. 2003 October; 13(5):551-7.) Animal cells andorganisms move, conduct cell divisions which serve to regenerate andmaintain tissues and circulating cell populations, grow in aconcurrently repetitive manner, contain a reserved germline set aside inembryogeny, have a low tolerance to genetic abnormalities, produceembryos complex and incomplete, and display essentially no asexualpropagation and have no cell wall. Plants respond by physiologicaladjustment, their cell divisions contribute to de novo formation oforgans all the way through to senescence, plant growth is serial,repetitive, and plastic, plants have no reserved germline, are moretolerant of genetic abnormalities, their embryos simple and complete,and plant cells are totipotent. Plant stem cells and seeds (plantgametes) are contemplated for use in this invention. Contrary to therarity of totipotent cells in animals, almost every cell formed by afungus can function as a “stem cell”. The multicellular fruiting bodiesof basidiomycete fungi consist of the same kind of filamentous hyphaethat form the feeding phase, or mycelium, of the organism, and visiblecellular differentiation is almost nonexistent (Money N P. Mushroom stemcells. Bioessays. 2002 October; 24(10):949-52).

The description is organized into the following sections: A. Adult stemcell extracts; B. Non-mammalian cell, egg and embryo extracts; C.Methods for preparing extracts; D. Epigenetic inhibitors; E. Enzymesinvolved in collagen synthesis and degradation; F. Ascorbic acid(vitamin C) as a cofactor in collagen synthesis; G. Iron as a cofactorin collagen synthesis; H. Topical delivery methods; I. Other deliverymethods; J. Additional components for extracts; K. Composition profiles;L. Topical application; M. Therapeutic uses.

A. Adult Stem Cell Extracts

In some embodiments, the present invention provides compositionscomprising adult stem cells or extracts prepared from adult stem cells.In some preferred embodiments, the cells or extracts are formulated fortopical application as described in more detail below. The adult stemcell is an undifferentiated (unspecialized) cell that is found in adifferentiated (specialized) tissue; it can renew itself and becomespecialized to yield specialized cell types of the tissue from which itoriginated. These precursor cells exist within the differentiatedtissues of the adult of all multicellular organisms in the animal,plant, protist and fungi kingdoms as a community of cells dispersedthroughout the tissue. Precursor cells derived from adults can bedivided into three categories based on their potential fordifferentiation. These three categories of precursor cells areepiblast-like stem cells, germ layer lineage stem cells, and progenitorcells. Precursor cells have been isolated from a wide variety oftissues, including, but not limited to, skeletal muscle, dermis, fat,cardiac muscle, granulation tissue, periosteum, perichondrium, brain,meninges, nerve sheaths, ligaments, tendons, blood vessels, bone marrow,trachea, lungs, esophagus, stomach, liver, intestines, spleen, pancreas,kidney, urinary bladder, and testis. Precursor cells can be releasedfrom the connective tissue compartments throughout the body bymechanical disruption and/or enzymatic digestion and have been isolatedfrom, but not limited to, newborns, adolescent, and geriatric mice, ratsand humans, and adult rabbits, dogs, goats, sheep, and pigs.

The first category of precursor cells, epiblast-like stem cells (ELSCs),consists of a stem cell that will form cells from all three embryonicgerm layer lineages. Stem cells from adult rats and stem cells fromadult humans can be released from the connective tissue compartmentsthroughout the body by mechanical disruption and/or enzymatic digestion.The stem cells from either adult rats or adult humans can bepreferentially slow frozen and stored at −80° C.±5° C. using 7.5%ultra-pure dimethyl sulfoxide. Fast thawing of stem cells from bothspecies from the frozen state to ambient temperature yields recoveryrates exceeding 98%. These cells in the undifferentiated state expressthe Oct-3/4 gene that is characteristic of embryonic stem cells. ELSCsdo not spontaneously differentiate in a serum free environment lackingprogression agents, proliferation agents, lineage-induction agents,and/or inhibitory factors, such as recombinant human leukemia inhibitoryfactor (LIF), recombinant murine leukemia inhibitory factor (ESGRO), orrecombinant human anti-differentiation factor (ADF). Embryonic stemcells spontaneously differentiate under these conditions. In contrast,ELSCs derived from both species remain quiescent unless acted upon byspecific proliferative and/or inductive agents and/or environment.

ELSCs proliferate to form multiple confluent layers of cells in vitro inthe presence of proliferation agents such as platelet-derived growthfactors and respond to lineage-induction agents. ELSCs respond tohepatocyte growth factor by forming cells belonging to the endodermallineage. Cell lines have expressed phenotypic markers for many discretecell types of ectodermal, mesodermal, and endodermal origin when exposedto general and specific induction agents.

The second category of precursor cells consists of three separate stemcells. Each of the cells forms cells of a specific embryonic germ layerlineage (ectodermal stem cells, mesodermal stem cells and endodermalstem cells). When exposed to general and specific inductive agents, germlayer lineage ectodermal stem cells can differentiated into, forexample, neuronal progenitor cells, neurons, ganglia, oligodendrocytes,astrocytes, synaptic vesicles, radial glial cells, and keratinocytes.

The third category of precursor cells present in adult tissues iscomposed of a multitude of multipotent, tripotent, bipotent, andunipotent progenitor cells. In solid tissues these cells are locatednear their respective differentiated cell types. Progenitor cells do nottypically display phenotypic expression markers for pluripotent ELSCs,such as stage specific embryonic antigen-4, stage-specific embryonicantigen-1 or stage-specific embryonic antigen-3, or carcinoembryonicantigen cell adhesion molecule-1. Similarly, progenitor cells do nottypically display phenotypic expression markers for germ layer lineagestem cells, such as nestin for cells of the ectodermal lineage orfetoprotein for cells of the endodermal lineage.

A progenitor cell may be multipotent, having the ability to formmultiple cell types. A precursor cell of ectodermal origin residing inthe adenohypophysisand designated the adenohypophyseal progenitor cellis an example of a multipotent progenitor cell. This cell will formgonadotrophs, somatotrophs, thyrotrophs, corticotrophs, and mammotrophs.Progenitor cells for particular cell lineages have unique profiles ofcell surface cluster of differentiation (CD) markers and unique profilesof phenotypic differentiation expression markers. Progenitor cells donot typically spontaneously differentiate in serum-free defined mediumin the absence of a differentiation agent, such as LIF or ADF. Thus,unlike embryonic stem cells which spontaneously differentiate underthese conditions, progenitor cells remain quiescent unless acted upon byproliferative agents (such as platelet-derived growth factor) and/orprogressive agents (such as insulin, insulin-like growth factor-I orinsulin-like growth factor-II).

Progenitor cells can regulate their behavior according to changingdemands such that after transplantation they activate from quiescence toproliferate and generate both new satellite cells and substantialamounts of new differentiated cells. For example, the contractile unitsof muscle are myofibers, elongated syncytial cells each containing manyhundreds of postmitotic myonuclei. Satellite cells are resident beneaththe basal lamina of myofibers and function as myogenic precursors duringmuscle regeneration. In response to muscle injury, satellite cells areactivated, proliferate, and differentiate, during which they fusetogether to repair or replace damaged myofibers. When satellite cellsare removed from their myofibers by a non-enzymatic physical titrationmethod, they retain their ability to generate substantial quantities ofnew muscle after grafting that they are not able to attain by enzymaticdigestion. Conventional enzymatic disaggregation techniques impairmyogenic potential. Collins and Partridge “Self-Renewal of the AdultSkeletal Muscle Satellite Cell” Cell Cycle 4:10, 1338-1341 (2005).

Accordingly, the present invention also contemplates the use ofnon-embryonic stem cells, such as those described above. In someembodiments, mesenchymal stem cells (MSCs) can be derived from marrow,periosteum, dermis and other tissues of mesodermal origin (See, e.g.,U.S. Pat. Nos. 5,591,625 and 5,486,359, each of which is incorporatedherein by reference). MSCs are the formative pluripotential blast cellsthat differentiate into the specific types of connective tissues (i.e.the tissues of the body that support the specialized elements;particularly adipose, areolar, osseous, cartilaginous, elastic, marrowstroma, muscle, and fibrous connective tissues) depending upon variousin vivo or in vitro environmental influences. Although these cells arenormally present at very low frequencies in bone marrow, various methodshave been described for isolating, purifying, and greatly replicatingthe marrow-derived mesenchymal stems cells in culture, i.e. in vitro(See also U.S. Pat. Nos. 5,197,985 and 5,226,914 and PCT Publication No.WO 92/22584, each of which are incorporated herein by reference).

Various methods have also been described for the isolation ofhematopoietic stem cells (See, e.g., U.S. Pat. Nos. 5,061,620;5,750,397; 5,716,827 all of which are incorporated herein by reference).It is contemplated that the methods of the present invention can be usedto produce lymphoid, myeloid and erythroid cells from hematopoietic stemcells. The lymphoid lineage, comprising B-cells and T-cells, providesfor the production of antibodies, regulation of the cellular immunesystem, detection of foreign agents in the blood, detection of cellsforeign to the host, and the like. The myeloid lineage, which includesmonocytes, granulocytes, megakaryocytes as well as other cells, monitorsfor the presence of foreign bodies in the blood stream, providesprotection against neoplastic cells, scavenges foreign materials in theblood stream, produces platelets, and the like. The erythroid lineageprovides the red blood cells, which act as oxygen carriers.

Accordingly, the present invention also contemplates the use of neuralstem cells, which are generally isolated from developing fetuses. Theisolation, culture, and use of neural stem cells are described in U.S.Pat. Nos. 5,654,183; 5,672,499; 5,750,376; 5,849,553; and 5,968,829, allof which are incorporated herein by reference. It is contemplated thatthe methods of the present invention can use neural stem cells toproduce neurons, glia, melanocytes, cartilage and connective tissue ofthe head and neck, stroma of various secretory glands and cells in theoutflow tract of the heart.

In some embodiments, extracts are prepared from the mammalian embryonicstem cells. In some embodiments, cells are washed in phosphate bufferedsaline (PBS) and in cell lysis buffer (100 mM HEPES, pH 8.2, 50 mM NaCl,5 mM MgCl₂, 1 mM dithiothreitol and protease inhibitors), sedimented at400 g, resuspended in 1 volume of cold cell lysis buffer and incubatedfor 30-45 min on ice to allow swelling. Cells are sonicated on ice in200-μl aliquots using a Labsonic-M pulse sonicator fitted with a 3-mmdiameter probe (B. Braun Biotech, Melsungen, Germany) until all cellsand nuclei are lysed. The lysate is sedimented at 15,000 g for 15 min at4° C. to pellet the coarse material. The supernatant is aliquoted,frozen in liquid nitrogen and can be stored for up to 9 months at −80°C. If necessary, extracts can be diluted with H₂O prior to use to adjustthe osmolarity to ˜300 mOsm (i.e., isotonicity).

In some embodiments, the adult stem cell extracts are used as is, whilein other embodiments, the extracts are formulated either alone or withother components as described in more detail below.

B. Non-Mammalian Cell, Egg and Embryo Extracts

In some embodiments, the compositions of the present invention utilizecell, egg and embryo extracts from vertebrates, including but notlimited to Superclass Gnathostomata (jawed vertebrates), Euteleostomi(bony vertebrates), Class Actinopterygii (ray-finned fishes), ClassSarcopterygii (lobe-finned fishes and terrestrial vertebrates),Tetrapoda (tetrapods), Amniota (amniotes), Synapsida (synapsids), ClassMammalia (mammals), Early Therapsida (early therapsids), Class Reptilia(reptiles), Anapsida (tortoises and turtles), Order Testudines(tortoises and turtles), Diapsida (birds, crocodiles, lizards, snakes,and relatives), Archosauria (birds and crocodiles), Order Crocodilia(caimans, crocodiles, and relatives), Lepidosauria (amphisbaenians,lizards, snakes, and tuataras), Order Rhynchocephalia (tuataras), OrderSquamata (amphisbaenians, lizards, and snakes), Class Amphibia(amphibians), Subclass Dipnoi (lungfishes), Actinistia, OrderCoelacanthiformes (coelacanths), Class Chondrichthyes (rays, sharks, andrelatives), Placodermi (armored fishes and placoderms), ClassCephalaspidomorphi, more preferably fish, shrimp, sea urchin oramphibian eggs or embryos. In some embodiments, unfertilized butactivated fish, shrimp, sea urchin or amphibian eggs are used. Thepresent invention is not limited to the use of any particular types ofeggs. Indeed, the use of a variety of eggs is contemplated, including,but not limited to eggs from Xenopus, shrimp, sea urchin, salmon, troutor zebrafish. In some embodiments, eggs are collected from maturefemales and spontaneously activate upon contact with water. In furtherembodiments, the eggs are washed in Ringer's saline. In someembodiments, the eggs are not from an avian species.

C. Preparation and Stabilization of Extracts and Fractions

Extracts of the present invention are prepared from any of the sourcesdescribed in section A-D. In some embodiments, the extracts are cellularextracts. Cellular extracts of the present invention are compositions ofdisrupted cells such as stem cells or eggs. The cells may be disruptedby a variety of methods, including, but not limited to, mechanicalshearing or blending, sonication, or osmotic lysis. In some embodiments,the cellular extracts are preferably further processed to yield acomposition that is substantially free of lipids naturally associatedwith the cells, such as cell membrane components. By substantially freeof lipids, it is meant that the cellular extract comprises less thanabout 1%, preferably less than about 0.5%, and more preferably less thanabout 0.1% of lipids that are naturally associated with the cells usedto make the cellular extract. In some embodiments, the extracts compriseless than about 1% and preferably less than 0.1% cholesterol orovalbumin. Accordingly, in some embodiments, the cellular extractcomprises carbohydrates, proteins, glycosylated or otherwise modifiedproteins, peptides, amino acids, RNA (mRNA, sRNA, miRNA, rRNA), DNA,water etc, and combinations thereof. In some embodiments, the cellularextracts can comprise small amounts of lipids naturally associated withthe cells, as well as nuclear components such as chromosomes, nucleicacids, and nuclear proteins. In some embodiments, the cellular extractis preferably a cytoplasmic extract or fraction prepared by removingnuclear, cell membrane and other water insoluble materials naturallyassociated with the cells. In some embodiments, these components areremoved by centrifugation or fractionation of the disrupted cells. Insome embodiments, the cellular extract is preferably an aqueous extractor fraction comprising water soluble cellular components such asproteins, mRNA, and carbohydrates.

A variety of methods may be used to prepare extracts. For example, insome embodiments, eggs are placed “dry” in a glass 15 ml centrifugetube, and crushed by sedimentation at 15,000 g for 15 min. This producesthree layers: a lipid top fraction, which is collected, aliquoted andfrozen; a middle cellular or cytoplasmic fraction, which is alsocollected, aliquoted and frozen; and a pellet fraction, which isdiscarded. In some embodiments, the cellular fraction or extractprimarily comprises contents of the cytoplasm. The cellular fraction isused as extract. In some embodiments, the cellular fraction may be usedin combination with a lipid fraction. The cytoplasmic fraction may becleared further by sedimentation at 50,000, 100,000 or 200,000 g toyield a further cellular extract which is primarily a water solubleextract fraction. Regardless of the fraction used, the extract can bediluted to about 300 mOsm with cell lysis buffer (see above), ifnecessary. Accordingly, in some preferred embodiment's, a water solubleextract prepared from eggs or embryos is utilized.

In other embodiments, the eggs are suspended in 0.5 volume of cell lysisbuffer and sonicated on ice until all eggs are lysed. The particulatematerial is sedimented at 15,000 g for 15 min at 4° C. The supernatantconstitutes the extract. As above, osmolarity can be adjusted to 300mOsm if needed. The extract can also be cleared as above.

In still other embodiments, the eggs are suspended in cell lysis bufferas in method 2. Eggs are lysed by Dounce homogenization using a glassmortar and pestle (Kontes, type A or B). The lysate is sedimented andtreated as described above.

In some preferred embodiments, the present invention providescompositions, either prepared from natural sources as described above orfrom artificial source materials, or a combination thereof. In someembodiments, the extracts are characterized as having an osmolarity offrom about 330 to 440, preferably about 350 mOsm. In some embodiments,the extracts have a pH of from about 5.0 to about 7.7, preferably a pHof about 6.5-7.0. In some embodiments, the extracts have a proteincontent of about 100 to 250 mg/ml, preferably about 160 to 190 mg/ml,and most preferably about 120 mg/ml. In some embodiments, thecompositions have a water content of about 20 to 90 percent waterweight/weight (w/w), preferably about 37 to 79% water w/w. In someembodiments, the extracts have a density of about 0.8 to about 1.4 g/ml,preferably about 1.1 g/ml. In some embodiments, the compositionscomprise trace elements including, but not limited to, calcium,phosphorus, zinc, copper and iron. In some embodiments, the compositionscomprise vitamins, including, but not limited to vitamins A, C, E,riboflavin, niacin, B 6, calcium pantothenate and B 12. In someembodiments, the present invention provides a fresh roe compositioncomprising: 2.7 to 3.4% protein; 3 to 5% carbohydrates; 1.0 to 1.7% fatsin the form of phospholipids, and 0.01 to 0.05% minerals in fresh roe,should be less fats and higher total protein in the extract), 37 to 79weight percent water. In some embodiments, the extracts further comprisea lipid fraction. In some embodiments, the lipid fraction comprises fromabout 60% to about 80% unsaturated fatty acids. In further embodiments,the compositions comprise phospholipids, including phosphatidyl cholines(lecithins) or as phosphatidyl ethanolamine (cephalins), and to a lesserextent inositol phosphatides, cerebrosides and sphingomyelines. In someembodiments, the lipid fraction is from about 0.1% to about 1%, 2%, 3%,4% or 5% of the total composition, while in other embodiments, thecompositions are substantially free or free of lipids.

In some embodiments, the artificial extracts are prepared from 1) water,2) any type of protein (BSA, albumine, vitellogenin, amino acidmixtures, etc.), 3) vitamins and minerals as described above, 4) saltsor osmoles to create osmolarity of approx 350 mOsm, 5) glycerol or otheragent to increase viscosity, 6) lipids such as lecithins, cephalins andother phospholipids, 7) carbohydrates, 8) growth factors such as FGF,EGF and IGF, 9) and chemo-attractants such as SLC/6Ckine/Exodus2/TCA4and CKbeta-11/MIP-3beta/ELC, 10) acid or base to adjust pH to 6.2-7.2,and 11) preservatives such as methyl paraben, propyl paraben, BHA orBHT.

In some embodiments, the eggs or extracts are treated to preventbacterial growth. The use of a variety of methods is contemplated. Insome embodiments, the following methods are combined. In someembodiments, unfertilized or fertilized eggs (e.g., fish or amphibianeggs) are treated prior to homogenization with a bactericidal orbacteriostatic agent. Preferred agents include, but are not limited to,iodine containing agents such as betadine, buffodine, andpovidone-iodine, and other agents such as novasan, sodium hypochlorite,bacitracin, polymyxin B sulfate, silver containing compounds such assilver sulfadiazine and silver nitrate, mafenide acetate, nystatin,gentamicin, neomycin. In other embodiments, the extracts are treatedpost-homogenization to prevent bacterial growth. In some embodiments,the extracts, such as the cellular extracts or cytoplasmic fractions,are treated by heating. In some embodiments, the extracts are heated toabout 37, 40, 50, 60, 70, 80 or 90 degrees Celsius for about 30 secondsor 1, 2, 5, 10, 20, 30, 60 or 120 minutes.

In some embodiments, the eggs or extracts are filtered, preferablythrough 0.22 or 0.45 um filters to remove bacteria. In some embodiments,before or after filtering, the extracts are treated by additionalcentrifugation (15 min-2 hrs) after heating the extract to 56° C. tospin down any bacteria present.

In other embodiments, eggs are washed in a sulfur-containing agent(e.g., calcium polysulfide or calcium thiosulfate (lime sulfur)) priorto preparation. In some embodiments, sulfur is added to the extracts toremove bacteria. In other embodiments, benzoyl peroxide is added to theextracts. In some embodiments, eggs are washed in 0.001% to about 0.2%by weight of a metal chlorite and sufficient acid to adjust the pH ofthe solution from about 2.2 to about 4.5 to remove bacteria. In furtherembodiments, the eggs and/or extract are placed in a vacuum drum andmixed with a natural solution containing salt, vitamin C or citric acid,and water to remove bacteria. In some embodiments, the eggs and/orextract are stirred, vortexed, sonicated, agitated or shaken with saltwater or liquid buffer to dislodge bacteria and vacuum filter off theliquid to remove bacteria. It will be possible to check bacterialcontent in the liquid and on the treated eggs for quality control. Insome embodiments, electrophoresis of the eggs and/or extract is used toremove bacteria. It is contemplated that such methods utilize theinfluences of electrical double layer, intensity of electrical field,electric density gradient, pH of the buffer solution, ionic strength ofbuffer solution, stage of growth of bacteria, and anion surface-activeagent upon the electrophoretic mobility of some species of bacteria.

In some embodiments, lipids are removed by treatments the homogenateprior to centrifugation or the extract after centrifugation. The use ofa variety of methods is contemplated. In some embodiments, lipids areremoved by filtering through fat-absorbing paper or filter by applying avacuum suction system to a container with a filter in the bottom, wherethe extract is placed in the container and suctioned through the filter.In some embodiments, lipids are removed by using an absorbent materialand an outer containment vessel. The extract is entered to a containerfilled with absorbent material through a pump and then recovered byapplying a vacuum. In some embodiments, lipids are removed with hollowfiber contraction systems and/or extraction solvents for removing lipidsfrom viscous fluids, where contact a fluid with an extraction solvent,which causes the lipids in the fluid to separate from the fluid orcauses lipids in the lipid-containing organisms to separate from thelipid-containing organism, using at least one hollow fiber contactor.

In some embodiments, the homogenates and extracts may be stabilized bythe addition of one or more stabilizing agents, such as a lipidstabilizing agent, or by packaging in a package designed to preventoxidation. In some embodiments, antioxidants such as vitamin E are addedto the extract to reduce rate of lipid oxidation. In some embodiments,the extracts are packaged in a container under an inert atmosphere. Insome embodiments, the extract is packaged to reduce rate of lipidoxidation in air-free containers such as aluminum coated bags (less than10 kg per bag for efficient removal of oxygen), or containers filledwith nitrogen to remove oxygen. In other embodiments, the extracts arepackaged in vacuum packed containers with a pump delivery system.

In some embodiments, extracts from stem cells, such as embryonic stemcells, are prepared in a like manner. In these embodiments, the stemcells are first disrupted and then centrifuged as above to removeinsoluble cellular debris. The stem cells generally comprise much lesslipid material, so the initial centrifugation yields two main fractions,a pellet and cellular fractions which primarily contains cytoplasmiccomponents. In some embodiments, cells, either a plate of cells or cellscollected from flasks or fermentors, are washed in ice cold PBS. When aplate of cells is utilized, the cells are scraped and transferred to anice cold centrifuge tube, such as an Eppendorf tube. In someembodiments, the cells are then pelleted and the supernatant is removed.The cells are then disrupted. In some embodiments, a hypotonic solutionis added to the cells in a volume of from about 1.5:1 to 3.0:1 ascompared to the cell pellet. A suitable hypotonic solution comprises 10mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl 3.33, 0.5 mM DTT, and 0.2 mMPMSF. In some embodiments, a 10% solution of Triton X is then added(about 1/20 volume) to the pellet and the pellet resuspended byvortexing. In some embodiments, the cells are then homogenized, forexample with a Dounce homogenizer or sonicated to further disrupt thecells. In some embodiment, the cellular debris is then pelleted bycentrifugation, for example 6,000 RPM at 4° C. for 30 seconds. Thesupernatant is then collected as the cellular extract.

In some embodiments where fish eggs are utilized, the fish eggs aretreated to prevent bacterial growth as described above. The fish eggsare then homogenized by subjecting the fish eggs to a pressuretreatment. In some embodiments, the eggs are subjected to a pressure offrom about 1 ton to about 100 tons, preferably about 5 tons to about 50tons, more preferably about 10 tons to about 30 tons and most preferablyabout 20 tons. In some embodiments, the pressure is applied via ahydropress. Suitable hydropresses are available from Speidel. In someembodiments, components of the homogenate are separated. In somepreferred embodiments, an aqueous cytoplasmic fraction is obtained thatcomprises protein, DNA, RNA, and other components as described in moredetail elsewhere herein. In some embodiments, the extracts comprise alipid component in addition to the water soluble components. In someembodiments, the extract is separated from the homogenate bycentrifugation. In some embodiments, the centrifugation is acontinuous-feed process facilitated by a separator. Suitable separatorsare available, for instance, from GEA Westfalia.

In some embodiments, the present invention provides processes forproducing an active fish egg fraction comprising milling the eggsbetween two or more surfaces disposed so as to cause crushing of theeggs as the eggs pass the surfaces. In some embodiments, at least one ofthe surfaces comprises cutting elements, for example knurls. In someembodiments, the mill is a roller mill comprising two or morecylindrical rollers. In some embodiments, each of the rollers comprisinga cutting surface, for example a knurled surface. In some embodiments,the eggs are placed in a hopper and fed to the milling surface(s). Inthe case of a roller mill, the eggs are passed between the rollers,causing the eggs to be crushed to form a homogenate. In someembodiments, the milling surfaces (e.g., the surfaces of rollers) arespaced apart to effectuate crushing of eggs passing the surfaces. Forexample, in some embodiments, the surfaces are separated by from 0.1 to5 mm, preferably about 0.5 to 2 mm. In some preferred embodiments, theprocesses further comprise separating an active fraction from said fishegg homogenate, wherein the active fraction comprises about 100 to 380mg/ml protein in an aqueous solution; about 0.1 to 10 mg/ml RNA; about0.1 to 5 mg/ml DNA and 0.1-10% lipids w/w. In some embodiments, theseparating of the active fraction is by a centrifugal force, asdescribed above.

In some embodiments, the cellular extracts described above, and mostpreferably the middle fractions, are further fractionated. A variety ofmethod may be used, including, but not limited to, FICOL gradients,gradient centrifugation, protein precipitation, freeze drying, columnchromatography, such as size exclusion chromatography and affinitychromatography, gel separation, high pressure liquid chromatagraphy,ChIP, and immunoprecipitation. It will be recognized that these fractionsteps yield corresponding fractions such as freeze dried fractions,affinity chromatography fractions, precipitated fractions, etc.

In some embodiments, the fractions are then combined with orresolubilized with components suitable for preparing compositions fortopical administration as described in more detail below.

D. Epigenetic Inhibitors

In some embodiments, the compositions of the present invention furthercomprise epigenetic inhibitors. In preferred embodiments, one or moreepigenetic inhibitors are combined with one or more of the cellularextracts described in Sections A-E. The present invention is not limitedto the use of any particular epigenetic inhibitors. Indeed, the use ofvariety of epigenetic inhibitors is contemplated, including, but notlimited to synthetic epigenetic inhibitors and epigenetic inhibitorsisolated or derived from natural sources. Examples of epigeneticinhibitors include, but are not limited to histone deacetylaseinhibitors, DNA methyltransferase inhibitors and some vitamins.

In some embodiments, the epigenetic inhibitors comprises a naturalextract containing butyrate or butyric acid made from natural foods suchas butter from animal fats or milk (e.g. cows milk or cheese), plantoils (e.g. Heracleum giganteum (cow parsnip) and Pastinaca sativa(parsnip)), or Kombucha tea (includes Butyric Acid as a result offermentation containing butyrate). Extract preparation may includefermentation by obligate anaerobic bacteria (e.g. Clostridium butyricum,Clostridium kluyveri, Clostridium pasteurianum, Fusobacterium nucleatum,Butyrivibrio fibrisolvens, Eubacterium limosum). Animal fat or plant oilproduct extracts may be prepared by chemical or physical processesinducing the liberation of butyric acid from the glyceride byhydrolysis. The extract could also be prepared by the fermentation ofsugar or starch in the natural foods by the addition of Bacillussubtilis, with calcium carbonate added to neutralize the acids formed.

In other embodiments, the epigenetic inhibitors comprise a naturalextract of red grapes containing the phytoalexin resveratrol, includingan extract from juice or fermented juice (wine) of red grapes. Extractscould be prepared by mechanical disruption of grapes, separation of theskin from the flesh and seeds, and either extracting phytoalexin bychemical or mechanical methods, or be prepared from fresh or fermentedgrape juice by chemical or physical methods including boiling,fractionation, affinity chromatography, freeze-drying or gel separation.

In other embodiments, the epigenetic inhibitors comprise a naturalextract containing Cyanocobalamin (vitamin B-12) made from organismscontaining enzymes required for the synthesis of B12 such as bacteriaand archaea, or natural products which harbor such B12 producingbacteria including meat (especially liver and shellfish), eggs, and milkproducts. Extracts can be prepared by chemical or physical methods suchas homogenization followed by fractionation, affinity chromatography,freeze-drying or gel separation.

In other embodiments, the epigenetic inhibitors comprise a naturalextract containing one or several variants of vitamin B, made fromeither potatoes, bananas, lentils, chilli peppers, tempeh, liver,turkey, tuna, nutritional yeast (or brewer's yeast), beer or marmite.Extracts can be prepared by chemical or physical methods such ashomogenization followed by e.g. fractionation, affinity chromatography,freeze-drying or gel separation.

In other embodiments, the epigenetic inhibitors comprise a naturalextract containing retinoids or retinoid precursors, made from eitheranimal sources (e.g. milk and eggs) which contain retinyl esters, orfrom plants (e.g. carrots, spinach) which contain pro-vitamin Acarotenoids. The extract may be modified by hydrolysis (animal sources)of retinyl esters to result in retinol, while plant extracts containingpro-vitamin A carotenoids can be cleaved to produce retinal(retinaldehyde), which can be further be reversibly reduced to produceretinol or it can be irreversibly oxidized to produce retinoic acid. Thebest described active retinoid metabolites are 11-cis-retinal and theall-trans and 9-cis-isomers of retinoic acid, which may be added to thisextract.

Examples of other DNA methyltransferase inhibitors include, but are notlimited to, 5-Azacytidine, 5-Aza-20-deoxycytidine,Arabinosyl-5-azacytidine, 5-6-Dihydro-5-azacytidine,5-Fluoro-20-deoxycytidine, EGX30P, Epigallocatechin-3-gallate, Green teapolyphenol, Hydralazine, MG98, Procainamide, Procaine, and Zebularine.Examples of other histone deacetylase inhibitors include, but are notlimited to Apicidin, Butyrates, Phenylbutyrate, m-Carboxycinnamic acidbishydroxamide (CBHA), Cyclic hydroxamic-acid-containing peptide 1(CHAP1), TSA-Trapoxin Hybrid, Depudecin Epoxide, Depsipeptide FR901228,Benzamidine, LAQ824, Oxamflatin, MGCD0103, PXD101, Pyroxamide, SubericBishydroxamic Acid (SBHA), Suberoylanilide Hydroxamic Acid (SAHA),Trichostatin A (TSA), Trapoxin A, and Valproic acid.

E. Enzymes Involved in Collagen Synthesis and Degradation

In some embodiments, the extracts described above (or components of theextracts) are in a composition further including, but not limited to theenzymes, procollagen peptidase, which form tropocollagen; hydroxylases,responsible for the step of hydroxylation of selected proline and lysineamino acids in the newly synthesized procollagen protein. Thehydroxylase enzymes require Vitamin C and Iron as cofactors. If apatient is Vitamin C deficient, then this reaction will not occur(Reference: Mussini E, Hutton J J, Udenfriend S. Collagen prolinehydroxylase in wound healing, granuloma formation, scurvy, and growth.Science 1967; 157:927-9).

F. Ascorbic Acid (Vitamin C) as a Cofactor in Collagen Synthesis

In some embodiments, the extracts described above (or components of theextracts) are in a composition further including ascorbic acid (VitaminC) on which the hydroxylation step in the formation of collagenintracellularly is dependent. Additionally, Vitamin C is beneficial forskin because deficiency causes scurvy, a serious and painful disease inwhich defective collagen prevents the formation of strong connectivetissue. Gums deteriorate and bleed, with loss of teeth; skin discolors,and wounds do not heal.

G. Iron as a Cofactor in Collagen Synthesis

In some embodiments, the extracts described above (or components of theextracts) are in a composition further including iron (Fe) on which thehydroxylation step in the formation of collagen intracellularly isdependent.

H. Other Delivery Methods

In some embodiments, the extracts described above (or components of theextracts) are formulated for delivery by a variety of methods. In someembodiments, the extracts described above are formulated for delivery toskin, gastrointestinal tractus, fat deposits, cartilage, bone,connective tissue, muscle or internal organs. In some embodiments, theextracts or components thereof are formulated for oral administrationwith or without suitable carriers such as starch, sucrose or lactose intablets, pills, dragees, capsules, solutions, liquids, slurries,suspensions and emulsions. In some embodiments, the oral deliveryvehicle comprises an enteric coating. In other embodiments, the extractsor components thereof are formulated for rectal administration as acapsule, cream, suppository or liquid. In some embodiments, the extractsof components thereof are injected by syringe to the peritoneal cavityor into internal organs or tissues. In some embodiments, the extracts orcomponents thereof are formulated for delivery an osmotic pump.

In still other embodiments, the extracts or components thereof aredelivered by microinjection, preferably via particle bombardment (i.e.,with a gene gun). Particle mediated gene transfer methods are known inthe art, are commercially available, and include, but are not limitedto, the gas driven gene delivery instrument descried in McCabe, U.S.Pat. No. 5,584,807, the entire contents of which are herein incorporatedby reference. This method involves coating the nucleic acid sequence ofinterest onto heavy metal particles, and accelerating the coatedparticles under the pressure of compressed gas for delivery to thetarget tissue. Other particle bombardment methods are also available.Generally, these methods involve depositing the extract or componentsthereof upon the surface of small, dense particles of a material such asgold, platinum, or tungsten. The coated particles are themselves thencoated onto either a rigid surface, such as a metal plate, or onto acarrier sheet made of a fragile material such as mylar. The coated sheetis then accelerated toward the target biological tissue. The use of theflat sheet generates a uniform spread of accelerated particles thatmaximizes the number of cells receiving particles under uniformconditions, resulting in the introduction of the nucleic acid sampleinto the target tissue. This invention contemplates the described use ofgene-gun to deliver extracts or components of extracts as defined above.

In still other embodiments, the embryonic stem cell, adult stem cell oregg extracts or components are microencapsulated (e.g., with collagen orglycosaminoglycans), formed into nanoparticles (e.g., lecithinencapsulated in an oil core), liposomes, microemulsions, ornanoemulsions, oil bodies, retinol molecular fluid films, unilamellarvesicles, multilamellar vesicles, preloaded spherical beads or sponges,elastic vesicles, etc.

I. Composition Profiles

In some embodiments the composition for topical and/or internalapplication is a combination of extracts with lipids and/or water and/orcarbohydrates and/or nucleic acids and/or proteins and/or signalingsubstances. In some embodiments the extract herein is composed of wholecells or a combination of lipids and/or carbohydrates and/or nucleicacids and/or proteins and/or signaling substances of the cells fromwhich the extract is made, or from synthetic and/or natural versions oflipids and/or carbohydrates and/or nucleic acids and/or proteins and/orsignaling substances. Signaling profiles include combinations of activesubstances released from cells which are contained in extracts of cells,and include synthetic and/or natural versions of these signalingsubstances added to extracts. Signaling substances contemplated includebut are not limited to growth factors, endorphins, hormones, amino acidtransmitters, immunoregulatory cytokines and other immunity-associatedfactors.

Growth factor-β1 orchestrates the biology of irradiated tissue as atissue level sensor of oxidative stress, and is integral to the cellularDNA damage response. Transforming growth factor-β5 (TGF-β5), a member ofthis signaling factor family found in amphibians, is expressed inregenerating blastemas formed under limb regeneration (King et al.,2003), and all mammalian isoforms of TGF-β are released locally fromvarious cells at sites of injury and are important in the control offibrosis and scarring during mammalian tissue repair. Manipulation ofspecific TGF-β isoforms is capable of producing scar-free healing ofwounds in mice (Ferguson and O'Kane, 2004). TGF-β1 is a potentimmunoregulatory cytokine involved in suppression of inflammation andregulatory T cell activity, resulting in immune tolerance (Chen andWahl, 2003). Studies on wound healing and immunosuppression in mammalsindicates that differential activity of TGF-β in regenerating amphibianlimb stumps may be involved suppression of fibrosis and establishingconditions permissive for blastema formation.

Transforming growth factor-alpha (TGF-alpha) and brain-derivedneurotrophic factor (BDNF) secreted in vitro from human pluripotent stemcells derived from embryonic germ cells, termed embryoid body-derived(EBD) cells, have the capacity to restore neurologic function in animalsby protecting host neurons from death and facilitate reafferentation ofmotor neuron cell bodies (Kerr D A, et al., Human embryonic germ cellderivatives facilitate motor recovery of rats with diffuse motor neuroninjury. J Neurosci. 2003 Jun. 15; 23(12):5131-40).

Fibroblast growth factors (FGFs) such as FGF-10 have been demonstratedto be of importance in regrowth of limbs in frogs (Christen and Slack,1997; Yokoyama et al., 2000).

The Pro-opiomelanocortin (POMC) precursor for a-melanocyte stimulatinghormone (α-MSH), endorphins, and several other peptide hormones, isexpressed in regeneration blastemas (King et al., 2003), in skin as wellas brain, pituitary, and other organs. POMC is a central importance inmodulating immune activity within skin, primarily due to the activity ofα-MSH (Luger et al., 1999). Paracrine release of α-MSH peptides exerts apotent immunomodulatory effect on immune cells. α-MSH inhibits all formsof inflammation against which it has been tested (Lipton et al., 1997)and localized production of α-MSH helps maintain optimal immune responseat specific sites in the skin (Paus et al., 2003). Expression of α-MSHcells of a blastoma would be expected to confer an anti-inflammatoryeffect potentially important for inhibiting fibrosis and regenerationnecessary for limb or tissue regrowth.

Thymosin-β4 is a thymic maturation factor that has also been shown topromote angiogenesis, keratinocyte migration and wound healing (Malindaet al., 1999). thymosin-β4 exerts potent anti-inflammatory activity andis secreted by macrophages and T lymphocytes of skin, gut and otherorgans in addition to the thymus (Young et al., 1999; Girardi et al.,2003). Thymosin-β4 is up-regulated in frog pseudoblastemas (King et al.,2003) and regenerating blastemas and activities of thymosin-β4 intissues of amputated limbs may include immunomodulation of theinflammatory response in addition to stimulation of epithelial migrationand other aspects of regeneration.

J. Additional Components

In some embodiments, the extracts or components thereof described aboveare combined with additional components. In some embodiments, theseadditional components enhance uptake, bioavailability or penetration ofthe extract components. In preferred embodiments, extract components maycontain natural or a mixture of synthetic components. The components maybe partially or totally synthetic. In some embodiments, the cell orextract or synthetic components made from substances identified in theextracts are mixed with a composition comprising water, sebaceous andepidermal lipids and cell extracts, proteins, and components thereof,preferably comprises about a 10% lipid fraction by weight, about a 10%protein fraction by weight, and about an 80% volatile fraction byweight.

In some embodiments, the compositions of the present invention furthercomprise lipids, preferably lipids having beneficial effects in skin.The lipids include but are not limited to omega-3 fatty acids, myristicacid, and stearidonic acid, and combinations thereof. In someembodiments, the omega-3 fatty acid is eicosapentaenoic acid (EPA),which has been shown to have a photoprotective and anti-aging effect onskin (Kim H H. et al. Photoprotective and anti-skin-aging effects ofeicosapentaenoic acid in human skin in vivo. J Lipid Res. 2006 May;47(5):921-30.) In other embodiments, the omega-3 fatty acid isdocosapentaenoic acid (DPA), which is beneficial for the cell membranein fibroblast skin cells. In other embodiments, the omega-3 fatty acidis docosahexaenoic acid DHA). In some embodiments, the lipids comprise acombination of one or more these omega-3 fatty acids. Stearidonic acidreduces skin redness. Myristic acid increases penetration of activesubstances into skin, specifically into fibroblasts (Eric R. Brown andPapasani V. Subbaiah. Differential effects of eicosapentaenoic acid anddocosahexaenoic acid on human skin fibroblasts. Lipids. 1994 December29(12): 803-913); Zulfakar M. H. et al. Enhanced topical delivery and exvivo anti-inflammatory activity from a betamethasone dipropionateformulation containing fish oil. J Inflammation Res. Volume 59 (1):1-88); Mittal A, et al. The effect of penetration enhancers onpermeation kinetics of nitrendipine in two different skin models. BiolPharm Bull 2008 September; 31(9):1766-72.).

In further embodiments, the compositions further comprise naturalmarine-derived fat-soluble vitamins such as Vitamin A and E. These arewell known to be beneficial for skin both as antioxidants and by directeffect on fibroblast health. Vitamin E is vital in protecting skin cellsfrom ultra violet light, pollution, drugs, and other elements thatproduce cell damaging free radicals (Riedel S B et al. Vitamin E analog,alpha-tocopherol ether-linked acetic acid analog, alone and incombination with celecoxib, reduces multiplicity of ultraviolet-inducedskin cancers in mice. Anticancer Drugs. 2008 February; 19(2):175-81).Vitamin A has been shown in many studies to prevent and reversecancerous changes in cells in some parts of the body, including theskin, and repairs skin damage caused by the sun (REFERENCE: Alberts D.et al. Safety and efficacy of dose-intensive oral vitamin A in subjectswith sun-damaged skin. Clinical Cancer Research 2004; 10:1875-80.).

In further embodiments the compositions comprise natural or syntheticproteins including but not limited to the protein vitellogenin, knownfor its rejuvenating properties. Vitellogenin is also present in honey,heralded for giving bees prolonged lives (Munch D, Amdam G V. Thecurious case of aging plasticity in honey bees. FEBS Lett. 2010 Apr.10). Ref 9) In further embodiments the composition contains natural orsynthetic peptides, herein defined as polymers formed from the linking,in a defined order, of α-amino acids; including but not limited to milkpeptides, ribosomal peptides, nonribosomal peptides, peptones andpeptide fragments. Peptides are believed to have a good effect on skinand wrinkles. Because peptides are so small, it is thought that they maymore easily penetrate the skin and yield their effects.

Vernix caseosa (vernix) is a naturally occurring skin protectant. Vernixis a lipid rich substance composed of sebum, epidermal lipids, anddesquamated epithelial cells that progressively covers the skin of thedeveloping fetus, completely surrounded by amniotic fluid, during thelast trimester of pregnancy. In some embodiment, the invention relatesto compositions where the lipid fraction preferably comprises componentsin vernix, i.e., lecithin and other phospholipids, squalene, waxes, waxesters, sterol esters, diol esters, triglycerides, free sterols and fourclasses of fatty acids ranging in chain length from C₁₂ to C₂₆ (straightchain saturated, straight chain unsaturated, branched chain saturated,and branched chain unsaturated). In preferred embodiments, the vernixlipid components are as follow, with the relative percentages indicated,squalene (9%), aliphatic waxes (12%), sterol esters (33%), diesters(7%), triglycerides (26%), free sterols (9%), other lipids (4%). Inadditional embodiments, the lipid composition is composed of lipids fromegg and/or fish roe with wound healing properties 30% of which arebarrier lipids (proteolipid matrix); cholesterol (1.1%, 52.8% ofbarrier), free fatty acids (0.6%, 27.7% of barrier), phospholipids(0.4%), ceramides (0.7%, 20.1% barrier). In another preferredembodiment, the protein fraction contains the protein components ofvernix, i.e., keratin, filaggrin, regulator proteins (e.g. EGF), andglutamine.

The fatty acids within the aliphatic waxes may be branched and thebranched fatty acids may be methylated. The protein fraction consists ofepidermally derived proteins, primarily keratin and filaggrin. Theprotein fraction also contains trace amounts in the range of aboutmicromolar to millimolar concentrations of regulatory proteins such asepidermal growth factor (EGF), and trace amounts of about nanomolar tomicromolar concentrations of surfactant protein such as Surfactant A andSurfactant B. The volatile fraction is primarily water. The rate ofevaporation of volatile components is relatively slow, presumably due toincreased energy requirements for the dissociation of hydrogen bonds andfor diffusion from the cellular component through the lipid component tochange water from the liquid to the gaseous state. In additionalpreferred embodiments, the composition contains mRNA contained in cellextracts, preferably stem cell extracts.

In some embodiments, the embryonic stem cell, adult stem cell or eggextracts or components are combined with phospholipids or otherlipophilic substances, palmitylmyristrates, dimethylsulfoxide (DMSO),chitosan, long chain organic polymers such as polysaccharides,non-aqueous solvents, beta-glucan, pH adjusting components, skinmetabolism inhibition agents, propylene glycol, butylenes glycol,polyethylene glycol, olive oil or other naturally occurring oils,dimethyl isosorbide, dimethylformamide, methyl salicylate, long chainoleic acid, mucopolysaccharides, and other agents.

In some embodiments, the additional agents include, but are not limitedto, ubiquitin, antimicrobial agents (alpha-defensins, LL37,beta-defensins, etc.), surfactant proteins from the collectin family(collecting associated protein A and D), nicotinamide and psoriacin.

In some embodiments, the additional agents include, but are not limitedto, vitamins, antioxidants, minerals, extracts, and chemical compoundssuch as alpha-tocopherol (vitamin E), melanin, vitamin C, provitamin A,retinyl proprionate, retinoic acid, Vitamin D3, Nicotinamide (vitaminB), Niacinaminde (Vit B3, exfoliates surface skin), d-panthenol (aids inskin repair of damage), vitamin A, hyaluronic acid, ceramides, Seaweed(algae) Mineral oil (paraffinum liquidium) Petrolatum GlycerinIsohexadecane Cirtus aurantifolia (lime) extract Microcrystalline wax(cera microcristallina) Lanolin alcohol Seamum indicium (sesame) seedoil, Eucalyptus globules (eucalyptus) leaf oil, Magnesium sulfate,Sesamum indicum (sesame) seeds, Medicago satvia (alfalfa) seeds,Helianthus annuus (sunflower) seeds, Prunus dulcis (powdered almonds),Sodium, Potassium, Copper, Calcium, Magnesium, zinc gluconate, Paraffin,Vitamin E succinate, Niacin, Beta-carotene, Decyl oleate, Aluminumdistearate, Octyuldodecanol, Citric acid, Cyanocobalamin, Magnesiumstearate, Panthenol, Limonene, Geraniol, Linalool, Hydroxycitronellal,Citronellol, Benzyl salicylate, Citral, Methylchloroisothiazoline,Methylisothiazolinone, Alcohol denat., Fragrance (parfum), Butyleneglycol, Byrospermum parkii (shea butter), Fish (pisces) cartilageextract, Polyethylene, Hydrogenated polyisobutene, Cyclopentasiloxane,Cetyl esters, Cetearyl alcohol, Malachite, Isostearyl neopentanoate,Polybutene, Sucrose, Silica, Tocotrienol, Cucumis satvius (cucumber)fruit extract, Centella asiatica (hydrocotyl) extract, Seamum indicium(sesame) seeds, Eucalyptus globules (eucalyptus) leaf oil, Medicagosatvia (alfalfa) seeds, Helianthus annuus (sunflower) seeds, Prunusdulcis (powdered almonds), Potassium, Copper, Calcium, Magnesium,Caffeine, Sodiumhyaluronate, Linoleic acidCholesteryl/behenyl/octyldodecyl lauroyl glutamate, Methyl glucosesesquisterate, Cholesterol, Dimethicone, Ocimum basilicum (basil),Mentha arvensis (wild mint), Acrylates/C10-30 alkyl acrylatecrosspolymer, Glyceryl distearate, Cetearyl glucoside, Steareth-10,Carbomer, Aminomethyl propanol, Limonene, Linalool, Benzyl salicylate,Disodium EDTA, BHT, Sodium dehydroacetate, Phenoxyethanol,Methylparaben, Titanium dioxide (CI 77891), C12-20 acid PEG-8 Ester,Hydrogenated vegetable oil, Petrolatum, Butylene Glycol, Glycerin,Acetylated Lanolin, Glycoproteins, Panax, Ginseng Root extract,Equisetum Arvense (Horsetail) Extract, Sodium carbomer, Beeswax (ceraalba), Cetyl phosphate, Polyperfluoromethylisoporpyl ether, Benzylalcohol, Linalool, Hydroxycitronellal, Alpha-isomethyl ionone, Amylcinnamal, Hexyl cinnamal, Verenia furfuracea (treemoss) extract,Geraniol, Benzyl benzoate, Bytulphenol methylpropional, Eugenol, Benzylsalicylate, Chlorphenesin, Phenoxyethanol, and Methylparaben.

In some embodiments, the compositions of the present invention areuseful for facilitating the delivery of active compounds via the skin.In some preferred embodiments, one or more active agents, such as aprotein, small organic compound, or one of the agents identified aboveare combined with the cytoplasmic fraction of, for example, a fertilizedor unfertilized amphibian or fish eggs. Cytoplasmic fractions and methodfor making such fractions are disclosed elsewhere in the application indetail. Accordingly, in some embodiments, the present invention providescompositions comprising a cytoplasmic fraction of amphibian or fish eggsand one or more active agents. In some embodiments, the presentinvention provides methods of facilitating the penetration of one ormore active agents into the skin, comprising providing a compositioncomprising a cytoplasmic extract from amphibian and/or fish eggs and oneor more active agents and contacting the skin of a subject with thecomposition. As described above, the composition can be preferably be anemulsion, salve, cream, gel, spray, aerosol, liquid, etc.

Exemplary proteins that can be active agents include, but are notlimited to, Alzheimer's amyloid peptide (Aβ), SOD 1, presenillin 1 and2, renin, α-synuclein, amyloid A, amyloid P, activin, anti-HER-2,bombesin, enkephalinase, protease inhibitors, therapeutic enzymes,α1-antitrypsin, mammalian trypsin inhibitor, mammalian pancreatictrypsin inhibitor, calcitonin, cardiac hypertrophy factor,cardiotrophins (such as cardiotrophin-1), CD proteins (such as CD-3,CD-4, CD-8 and CD-19), CFTR, CTNF, DNase, human chorionic gonadotropin,mouse gonadotropin-associated peptide, cytokines, transthyretin, amylin,lipoproteins, lymphokines, lysozyme, a growth hormone (including humangrowth hormone), bovine growth hormone, growth hormone releasing factor,parathyroid hormone, thyroid stimulating hormone, growth factors,brain-derived neurotrophic growth factor, epidermal growth factor (EGF),fibroblast growth factor (such as α FGF and β FGF), insulin-like growthfactor-I and -II, des(1-3)-IGF-I (brain IGF-I), insulin-like growthfactor binding proteins, nerve growth factor (such as NGF-β),platelet-derived growth factor (PDGF), vascular endothelial growthfactor (VEGF), receptors for growth hormones or growth factors,transforming growth factor (TGF) (such as TGF-α, TGF-β1, TGF-β2, TGF-β3,TGF-β4 or TGF-β5), neurotrophic factors (such as neurotrophin-3, -4, -5,or -6), gelsolin, glucagon, kallikreins, mullerian-inhibiting substance,neurotrophic factors, p53, protein A or D, prorelaxin, relaxin A-chain,relaxin B-chain, rheumatoid factors, rhodopsin, a serum albumin (such ashuman serum albumin), inhibin, insulin, insulin chains, insulin A-chain,insulin β-chain, insulin receptor, proinsulin, luteinizing hormone,integrin, interleukins (ILs) (such as IL-1 to IL-10, IL12, IL-13),erythropoietin, thrombopoietin, fibrillin, follicle stimulating hormone,clotting factors (such as factor VIIIC, factor IX, tissue factor, andvon Willebrands factor, anti-clotting factors (such as Protein C, atrialnaturietic factor, lung surfactant), a plasminogen activator (such ashuman tissue plasminogen activator or urokinase), thrombin, tumornecrosis factor-α or β, α-ketoacid dehydrogenase, addressins, bonemorphogenetic proteins (BMPs), collagen, colony stimulating factors(CSFs) (such as M-CSF, GM-CSF and G-CSF), decay accelerating factor,homing receptors, interferons (such as interferon-α, -β and -γ),keratin, osteoinductive factors, PRNP, regulatory proteins, superoxidedismutase, surface membrane proteins, transport proteins, T-cellreceptors, viral antigens such as a portion of the AIDS envelope,immunoglobulin light chain, antibodies, antibody fragments (such assingle-chain Fv fragment (scFv), single-chain antibody (scAb), F_(AB)antibody fragment, diabody, triabody, fluorobody), antigens such asgp120(IIIb) immunotoxins, atrial natriuretic peptide, seminal vesicleexocrine protein, β2-microglobulin, PrP, precalcitonin, ataxin 1, ataxin2, ataxin 3, ataxin 6, ataxin 7, huntingtin, androgen receptor,CREB-binding protein, gp120, p300, CREB, AP1, ras, NFAT, jun, fos,dentaorubral pallidoluysian atrophy-associated protein, a microbialprotein (e.g., maltose binding protein, ABC transporter, glutathione Stransferase, thioredoxin, β-lactamase), green fluorescent protein, redfluorescent protein, or derivatives or active fragments or geneticvariants of any of the peptides listed above.

Examples of small organic compounds include, but are not limited to,non-steroidal anti-inflammatory drugs (NSAIDS)(the NAIDS can, forexample, be selected from the following categories: (e.g., propionicacid derivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams)); steroidalanti-inflammatory drugs including hydrocortisone and the like;antihistaminic drugs (e.g., chlorpheniranune, triprolidine); antitussivedrugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane);antipruritic drugs (e.g., methidilizine and trimeprizine);anticholinergic drugs (e.g., scopolamine, atropine, homatropine,levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine,meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); minoxidil; antiarrhythmic drugs (e.g., propanolol,procainamide, disopyraminde, quinidine, encainide); P-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, amrinone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone,isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chlorprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); PDE5 inhibitorssuch as Viagra® or Cialis®; tranquilizer and sedative drugs (e.g.,chlordiazepoxide, benacytyzine, benzquinamide, flurazapam, hydroxyzine,loxapine and promazine); antipsychotic drugs (e.g., chlorprothixene,fluphenazine, haloperidol, molindone, thioridazine and trifluoperazine);antimicrobial drugs (antibacterial, antifungal, antiprotozoal andantiviral drugs).

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, triclosan, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isothionate, metronidazole;pentamidine, gentamycin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmycin, paromomycin,streptomycin, tobramycin, miconazole, and amanfadine.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, a-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine).

The compositions can also comprise hormones (e.g., medroxyprogesterone,estradiol, leuprolide, megestrol, octreotide or somatostatin); musclerelaxant drugs (e.g., cinnamedrine, cyclobenzaprine, flavoxate,orphenadrine, papaverine, mebeverine, idaverine, ritodrine,dephenoxylate, dantrolene and azumolen); antispasmodic drugs;bone-active drugs (e.g., diphosphonate and phosphonoalkylphosphinatedrug compounds); endocrine modulating drugs (e.g., contraceptives (e.g.,ethinodiol, ethinyl estradiol, norethindrone, mestranol, desogestrel,medroxyprogesterone), modulators of diabetes (e.g., glyburide orchlorpropamide), anabolics, such as testolactone or stanozolol,androgens (e.g., methyltestosterone, testosterone or fluoxymesterone),antidiuretics (e.g., desmopressin) and calcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progenstogens, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful active compounds include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H₂ antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

In some embodiments, components of the extract may act as chemotaxants.Mesenchymal stem cells and fibrocytes circulates in the blood stream andin case of skin wound they penetrate the wound area where they candifferentiate to skin cells like fibroblasts, keratinocytes, pericytes,adipose and endothelial cells. Chemotaxants in the extract may act asligands for the CCR7 involved in attractin immune cells and dendriticcells and may include SLC/6Ckine/Exodus2/TCA4 andCKbeta-11/MIP-3beta/ELC

K. Topical Application

In some embodiments, the extracts described above (or components of theextracts) are formulated for topical delivery. General formulations fortopical delivery are described in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing, p. 1288-1300 [1990]. Accordingly, in someembodiments, the extracts are formulated as a water based gel or paste,ointment, cream (anhydrous or hydrous), lotion (anhydrous or hydrous),emulsion, spray, solution, aerosol, stick (solid cream), liquid bandaid, powder, inhalation spray, nasal spray, basal drops, cheek drops,sublingual drops, eye drops or sprays, ear drops or sprays, andtransdermal patches.

It is contemplated that the compositions for topical application finduse for both cosmetic and therapeutic purposes. In some embodiments, itis contemplated that the compositions described above are applieddirectly to the skin or other epithelial or epidermal surfaces of thebody. The compositions may be applied one, two, three or more times eachday as is appropriate for the indication. The amount applied is notgenerally important, but generally a composition comprising from about0.001 μg to 10 grams of the extract (or components thereof) may beapplied to a given surface of the body. As described above, thecomposition may comprise other components such as adjuvants, carriers,other active ingredients, etc.

In some embodiments, the invention relates to compositions that includepreservatives and antioxidants (including vitamins) to prevent productdeterioration preferably trisodium and tetrasodium edetate (EDTA) andtocopherol (vitamin E). In further embodiments the composition containsantimicrobials to fight bacteria preferably butyl, propyl, ethyl, andmethyl parabens, DMDM hydantoin, methylisothiazolinone phenoxyethanol(also rose ether fragrance component), quaternium-15. In furtherembodiments, the composition contains thickeners and waxes used in stickproducts such as lipsticks and blushers preferably candelilla, carnauba,and microcrystalline waxes carbomer and polyethylene—thickeners. Infurther embodiments, the composition contains solvents to dilutepreferably butylene glycol and propylene glycol, cyclomethicone(volatile silicone), ethanol (alcohol) and glycerin. In furtherembodiments, the composition contains emulsifiers to break up and refinepreferably glyceryl monostearate (also pearlescent agent), lauramide DEA(also foam booster) and polysorbates. In some embodiments, thecompositions contain color additives—synthetic organic colors derivedfrom coal and petroleum sources preferably D&C Red No. 7 Calcium Lake(and other dyes that do not dissolve in water), iron oxides, mica(iridescent), and aminophenols. In further embodiments, the compositionscontain pH adjusters to stabilize or adjust acids and bases preferablyammonium hydroxide—in skin peels and hair waving and straightening,citric acid—adjusts pH, and triethanolamine—pH adjuster used mostly intransparent soap. In further embodiments, the compositions containsagents preferably magnesium aluminum silicate—absorbent, anti-cakingagent, silica (silicon dioxide)—absorbent, anti-caking, abrasive, sodiumlauryl sulfate—detergent, stearic acid—cleansing, emulsifier, talc(powdered magnesium silicate)—absorbent, anti-caking, and zincstearate—used in powder to improve texture, lubricates.

The composition includes the recited components and combinations thereofin a total amount of about 0.5 to 50 grams per liter, preferably about 3to 10 grams per liter, although higher or lower concentrations arepermissible. Such compositions being in the form of an emulsion, cream,salve or the like, the active materials being admixed with water,alkylene glycols, various oils natural and synthetic, petrolatum,preservatives, coloring agents, perfumes, and like ingredientsconventional in the cosmetic arts.

The composition can be applied to the face, eyelids or other body partsin an amount varying with the individual. About 0.01 to 1,advantageously about 0.02 to 0.75 and preferably about 0.3 to 0.5, gramsper cm² has been found useful but more or less can be used. Theapplication can be once weekly or more often, even several times a day.

In accordance with the compositions and method of the present invention,the egg, embryo or stem cell extracts of the present invention may beadministered in the form of a pharmaceutical composition additionallycomprising a pharmaceutically acceptable carrier. One skilled in the artwill appreciate that suitable methods of administering the extractcompositions to an animal, such as a mammal, are available and, althoughmore than one method can be used to administer a particular composition,a particular method and dosage can provide a more immediate and moreeffective reaction than others. Pharmaceutically acceptable carriers arealso well known to those skilled in the art. The choice of carrier willbe determined, in part, both by the particular composition and by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of the pharmaceuticalcompositions of the present invention.

In some preferred embodiments, the formulations of this invention aredesigned for topical administration. Typical of such formulations areointments, creams, and gels.

Ointments generally are prepared using either (1) an oleaginous base,i.e., one consisting of fixed oils or hydrocarbons, such as whitepetrolatum or mineral oil, or (2) an absorbant base, i.e., oneconsisting of an anhydrous substance or substances which can absorbwater, for example, anhydrous lanolin. Customarily, following formationof the base, whether oleaginous or absorbent, the active ingredient(e.g., salmon egg extract or stem cell extract) is added in an amountaffording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internalphase), comprising typically fixed oils, hydrocarbons, and the like,such as waxes, petrolatum, mineral oil, and the like, and an aqueousphase (continuous phase), comprising water and any water-solublesubstances, such as added salts. The two phases are stabilized by use ofan emulsifying agent, for example, a surface active agent, such assodium lauryl sulfate; hydrophilic colloids, such as acacia colloidalclays, veegum, and the like. Upon formation of the emulsion, the activeingredient (e.g., salmon egg extract or stem cell extract) customarilyis added in an amount to achieve the desired concentration.

Gels comprise a base selected from an oleaginous base, water, or anemulsion-suspension base, such as described above. To the base is addeda gelling agent which forms a matrix in the base, increasing itsviscosity. Examples of gelling agents are hydroxypropyl cellulose,acrylic acid polymers, and the like. Customarily, the active ingredient(IGF-II) is added to the formulation at the desired concentration at apoint preceding addition of the gelling agent.

Serums may be watery or thicker liquids, often (but not always) clear incolor. Serums are water based making them light in consistency. They areeasily and quickly absorbed into the skin and provide an excellent wayto deliver topical ingredients including Vitamin C, peptides, alphahydroxy acids, retinols. Serums may be layered under other serums aswell as creams or lotions making them a very flexible product toincorporate into your skin care regimen. Serums are tolerated well byall skin types as long as the individual is not sensitive to any of theingredients. Serums may include glycerol or glycerine. The amount ofextract incorporated into the formulation of this invention is notcritical; the concentration should only be in a range sufficient topermit ready application of the formulation to the wound area in anamount which will deliver the desired amount of extract.

The customary amount of formulation to be applied will depend uponconcentration of the active ingredient in the formulation. In someembodiments, the amount of protein in the extract is determined. Then, aspecific amount of the extract is included in the pharmaceuticallyacceptable carrier based on the amount of protein. Generally, theformulation will be applied to the wound in an amount affording fromabout 0.1 to about 500 μg of protein per cm² of skin. Preferably, theapplied amount of protein will range from about 1 to about 300 μg/cm²,more preferably, from about 5 to about 200 μg/cm². In other embodiments,a specific volume of extract is added to the pharmaceutically acceptablecarrier. Accordingly, in some embodiments, the compositions of thepresent invention comprise on a volume/volume basis (volume of extractand volume of pharmaceutically acceptable carrier), for example, fromabout 0.001 to 50% extract, about 0.01 to 50% extract, about 0.1 to 50%extract, about 0.001 to 10% extract, about 0.01 to 10% extract, about0.1 to 10% extract, about 0.001 to 5% extract, about 0.01 to 5% extract,about 0.1 to 5% extract, about 0.001 to 4% extract, about 0.01 to 4%extract, about 0.1 to 4% extract, about 0.001 to 2% extract, about 0.01to 2% extract, about 0.1 to 2% extract, about 0.001 to 1% extract, about0.01 to 1% extract, or about 0.1 to 1% extract.

The present invention may be formulated as necessary with additives usedcommonly in the pharmaceutical sciences, such as surfactants, oils andfats, polyhydric alcohols, lower alcohols, thickening agents, UVabsorbents, light scattering agents, preservatives, antioxidants,antibiotics, chelating agents, pH regulators, flavoring agents, pigmentsand water.

Examples of surfactants include polyoxyethylene (hereinafter abbreviatedas POE-branched alkyl ethers such as POE-octyldodecyl alcohol andPOE-2-decyltetradecyl alcohol, POE-alkyl ethers such as POE-oleylalcohol ether and POE-cetyl alcohol ether, sorbitan esters such assorbitan monooleate, sorbitan monoisostearate and sorbitan monolaurate,POE-sorbitan esters such as POE-sorbitan monooleate, POE-sorbitanmonoisostearate and POE-sorbitan monolaurate, fatty acid esters ofglycerol such as glyceryl monooleate, glyceryl monostearate and glycerylmonomyristate, POE-fatty acid esters of glycerol such as POE-glycerylmonooleate, POE-glyceryl monostearate and POE-glyceryl monomyristate,POE-dihydrocholesterol ester, POE-hardened castor oil, POE-hardenedcastor oil fatty acid esters such as POE-hardened castor oilisostearate, POE-alkylaryl ethers such as POE-octylphenol ether,glycerol esters such as glycerol monoisostearate and glycerolmonomyristate, POE-glycerol ethers such as POE-glycerol monoisostearateand POE-glycerol monomyristate, polyglycerol fatty acid esters such asdiglyceryl monostearate, decaglyceryl decastearate, decaglyceryldecaisostearate and diglyceryl diisostearate and other nonionicsurfactants; potassium salts, sodium salts, diethanolamine salts,triethanolamine salts, amino acid salts and other salts of higher fattyacids such as myristic acid, stearic acid, palmitic acid, behenic acid,isostearic acid and oleic acid, the above alkali salts of ethercarboxylic acids, salts of N-acylamino acids, N-acylsalconates, higheralkylsulfonates and other anionic surfactants; alkylamine salts,polyamine, aminoalcohol fatty acids, organic silicone resin, alkylquaternary ammonium salts and other cationic surfactants; and lecithin,betaine derivatives and other amphoteric surfactants.

Examples of oils and fats include vegetable oils and fats such ascastor-oil, olive oil, cacao oil, camellia oil, coconut oil, wood wax,jojoba oil, grape seed oil and avocado oil; animal oils and fats such asmink oil and egg yolk oil; waxes such as beeswax, whale wax, lanolin,carnauba wax and candelilla wax; hydrocarbons such as liquid paraffin,squalene, microcrystalline wax, ceresine wax, paraffin wax and vaseline;natural or synthetic fatty acids such as lauric acid, myristic acid,stearic acid, oleic acid, isostearic acid and behenic acid; natural orhigher alcohols such as cetanol, stearyl alcohol, hexyldecanol,octyldecanol and lauryl alcohol; and esters such as isopropyl myristate,isopropyl palmitate, octyldodecyl myristate, octyldodecyl oleate andcholesterol oleate.

Examples of polyhydric alcohols include ethylene glycol, polyethyleneglycol, propylene glycol, 1,3-butyrene glycol, 1,4-butyrene glycol,dipropylene glycol, glycerol, diglycerol, triglycerol, tetraglycerol andother polyglycerols, glucose, maltose, maltitose, sucrose, fructose,xylitose, sorbitol, maltotriose, threitol and erythritol.

Examples of thickening agents include naturally-occurring high molecularsubstances such as sodium alginate, xanthene gum, aluminum silicate,quince seed extract, gum tragacanth, starch, collagen and sodiumhyaluronate; semi-synthetic high molecular substances such as methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose, solublestarch and cationized cellulose; and synthetic high molecular substancessuch as carboxyvinyl polymer and polyvinyl alcohol.

Examples of UV absorbents include p-aminobenzoic acid, 2-ethoxyethylp-methoxycinnamate, isopropyl p-methoxycinnamate,butylmethoxybenzoylmethane,glyceryl-mono-2-ethylhexanoyl-di-p-methoxybenzophenone, digalloyltrioleate, 2,2′-dihydroxy-4-methoxybenzophenone,ethyl-4-bishydroxypropylaminobenzoate,2-ethylhexyl-2-cyano-3,3′-diphenyl acrylate, ethylhexylp-methoxycinnamate, 2-ethylhexyl salicylate, glyceryl p-aminobenzoate,homomethyl salicylate, methyl o-aminobenzoate,2-hydroxy-4-methoxybenzophenone, amyl p-dimethylaminobenzoate,2-phenylbenzoimidazole-5-sulfonic acid and2-hydroxy-4-methoxybenzophenone-5-sulfonic acid.

Examples of preservatives include benzoates, salicylates, sorbates,dehydroacetates, p-oxybenzoates, 2,4,4′-trichloro-2′-hydroxydiphenylether, 3,4,4′-trichlorocarbanilide, benzalkonium chloride, hinokitiol,resorcinol and ethanol.

Examples of antioxidants include tocopherol, ascorbic acid,butylhydroxyanisole, dibutylhydroxytoluene, nordihydroguaiaretic acidand propyl gallate.

Examples of chelating agents include sodium edetate and sodium citrate.

Examples of antibiotics include penicillin, neomycin, cephalothin,potassium permanganate, selenium sulfide, erythromycin, bacitracin,tethacyclin, chloramphenicol, vancomycin, nitrofurantoin, acrisorcin,chlorodontoin, and flucytosine.

Some of these additives function to enhance the efficacy of thecomposition by increasing the stability or percutaneous absorbability ofthe essential components of the present invention.

Also, any dosage form is acceptable, whether in solution, emulsion,powder dispersion, or others. Applicability is wide, includingfundamental dosage forms such as lotions, emulsions, creams and gels.

In addition to those stated above, suitable vehicles, carriers andadjuvants include water, vaseline, petrolatum, mineral oil, vegetableoil, animal oil, organic and inorganic waxes, polymers such asxanthanes, gelatin, cellulose, collagen, starch, kaolin, carrageenan,gum arabic, synthetic polymers, alcohols, polyols, and the like. Thecarrier can also include sustained release carrier such as lypizomes,microsponges, microspheres, or microcapsules, aqueous base ointments,water in oil or oil in water emulsions, gels or the like.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse over a reasonable time frame. The dose will be determined bythe strength of the particular compositions employed and the conditionof the person. The size of the dose and the frequency of applicationalso will be determined by the existence, nature, and extent of anyadverse side effects that may accompany the administration of aparticular composition.

L. Uses

In some embodiments, the cell or extract compositions are useful forhydration (i.e., treating intravascular dehydration and edema in awounds), waterproofing (i.e., compensate for hypovolemia in wounds),guarding against infection (i.e., protecting wound against infections),protection against oxidation (i.e., prevention of oxygen-free radicalproduction during inflammatory reactions of ischemic tissue), woundhealing (i.e., increased metabolism to aid in hypoxic conditionsespecially of burned skin or cells in anaerobic metabolism). In somepreferred embodiments, the compositions are odorless (i.e.,characterized by an absence of volatile carbon or nitrogen containingcompounds).

In some embodiments, the invention relates to methods of using aplurality of compositions. In preferred embodiments, a first cream isused to loosen and/or dissolve cars by collagen dissolving agents oracids, such as lactic acid. A second cream with extracts or componentsthereof as well as other wound healing substances as described herein.In another preferred embodiment a laser, chemical peel, razor, acid,freezing, exfoliant and/or abrasive is used to remove scars or wrinklesfollowed by application of a cream with extracts or components thereofas well as other wound healing substances as described herein.

In some embodiments, the invention relates to a first compositionpreferably a cream that slows wound healing, reduces inflammation,and/or reduces scab formation. This composition is applied for severaldays. In preferred embodiments, the composition comprises a combinationof one or more of anti-inflammatory agents, antihistamines, an extractcomponent or components capable of dampening neutrophil migration and/orproliferation, an extract component or components the stimulatemacrophages, phospholipases, arachidonic acid. In further embodiments,there is a water, lipid, protein content that provides vernix propertiesin the composition. In further embodiments, the components in thecomposition reduce activity of fibrogen cytokines. Preferably, the firstcomposition is applied for about 1 to 3 days.

In another embodiment, the invention relates to a second compositionpreferably a cream that heals wounds by stimulating needed cells.Preferably this composition is applied to a subject after the firstcomposition is applied. Preferably, the second composition is appliedfor about 3 to 14 days. This second composition contains components ofcell and cell extracts that regulate collagenases, activateplasminogenases for clot dissolution, stimulate epithelializaiton,(i.e., migration, proliferation, dedifferentiation, redifferentiation),activate fibronectin and fibroblast growth factors, stimulateangiogenesis, reduce activity of fibrogenic cytokines and regulate genessuch as TP53.

In another embodiment, the invention relates to a third compositionpreferably a cream. Preferably this composition is applied to a subjectafter the application of the second composition. This third compositionfunctions to control collagen remodeling by collagen synthesis anddestruction preferably by collegenases and metalloproteins andpreferably collagan I and inactivate fibronectin, hyaluroinic acid andglycosaminoglycans, and dehydrate swelling. The third composition ispreferably applied for about one to six weeks, following the applicationof the firs and second compositions. In some embodiments, a matrix isprovided, such as a chitosan matrix, biodegradable polymer matrix,collagen matrix, or liquid band aid.

In some embodiments, the cell and/or extract composition is dispersed ina biocompatible liquid was applied to a physiologically acceptablesupport structure in a liquid state to form a film. A film is definedherein as a surface and/or interfacial covering, in either a liquid or asolid state, with temperature-dependant properties. Film-formingtechniques include but are not limited to spraying, extruding, blowing,pouring, evaporating, coating and painting. The dispersion is presentedas droplets that coalesce to form a film upon encountering the support.

In an alternate embodiment, a preformed film is applied to a support.The physiologically acceptable support structure is one that canwithstand sterilization, preferably by standard sterilization techniquesknown to one skilled in the art such as exposure to gamma radiation,autoclaving, and so on. The support structure is not limited to aparticular composition or configuration and, depending upon its use, mayor may not be sterilized and may take various forms.

In another embodiment, the film is used to enhance skin cell maturationand may be applied to structures such as filters, membranes, beads,particles, and so on. Similarly, the support structure is not limited toa particular state of matter and may be a solid, a semi-solid, a gel andso on. In one embodiment, the support consists of a nylon monofilamentinterpositional surfacing material such as Interfaces pads (WinfieldLaboratories, Inc., Dallas Tex.), Biobrane II® (Sterling Drug Inc., NewYork, N.Y.) or circular nylon filters of suitable porosity (MicronSeparations Inc., Westboro, Mass.). Other support materials, however,could also be used to practice the invention.

In another embodiment, the film is used to treat or prevent injury dueto substance exposure or trauma, and may be applied to various materialsfor placement either in direct contact or indirect contact with anexposed skin site. The skin site may be intact (e.g., normal skin) ormay be compromised, defined as skin that is damaged or that lacks atleast some of the stratum corneum (e.g., skin damaged by exposure to theagent in question, another agent, the presence of a pathologicalcondition such as a rash or contact dermatitis, a physical trauma suchas a cut, wound, or abrasion, a underdeveloped skin such as occurs in apreterm infant, conditions in which either all or part of the epidermisis exposed, conditions in which part of the dermis has been removed suchas partial thickness wounds encountered in resurfacing procedures suchas chemical peels, dermabrasions, and laser resurfacing, etc.).

The support structure may be permeable to physical and/or chemicalagents, and may take a variety of forms, depending upon its purpose andthe extent of the area requiring dressing or treatment. The film may beapplied to various synthetics such as thermoplastic films, blown filmsand breathable films, and various natural and synthetic fabriccompositions such as woven, non-woven, spun, and stitched fabrics. Theinvention may be used in a variety of products, examples of whichinclude wound dressings and coverings such as bandages, tapes, gauze,adhesive products applied for a short or long term to the skin, ostomycare products, hospital pads such as incontinent pads, absorbent pads,and examination pads, disposable and cloth diapers, and feminine hygieneproducts such as intralabial devices.

In some embodiments, the invention relates to regeneration of thefunction of skin with a desired cosmetic appearance and the preventionof skin damage. In further embodiments, early scar formation isprevented by application of a scar prevention composition when the woundis formed. In further embodiments, stimulating the rejuvenation andregeneration of stressed and aging skin prevents wrinkle formation. Infurther embodiments, the product is applied intermittently to slow thecontinual damage process that occurs as skin ages.

The skin has two main layers, the epidermis and dermis. Below these is alayer of subcutaneous (‘under the skin’) fat. The outer surface of theskin is the epidermis, which itself contains several layers, the basalcell layer, the spinous layer, the granular cell layer, and the stratumcorneum. The deepest layer of the epidermis is the basal cell layer.Here cells are continually dividing to produce plump new skin cells.These cells move towards the skin surface, pushed upward by the dividingcells below them. Blood vessels in the dermis, which is below the basalcell layer, supply nutrients to support this active growth of new skincells. As the basal cells move upwards and away from their blood supply,their cell content and shape change. Cells above the basal cell layerbecome more irregular in shape and form the spinous layer. Above this,cells move into the granular layer. Being distant from the blood supplyin the dermis, the cells begin to die and accumulate a substance calledkeratin.

The stratum corneum (‘horny layer’) is the top layer of the epidermis—itis the layer of the skin that we see from the outside. Cells here areflat and scale-like (‘squamous’) in shape. These cells are dead, containa lot of keratin and are arranged in overlapping layers that impart atough and waterproof character to the skin's surface. Dead skin cellsare continually shed from the skin's surface. This is balanced by thedividing cells in the basal cell layer, thereby producing a state ofconstant renewal. Also in the basal cell layer are cells that producemelanin. Melanin is a pigment that is absorbed into the dividing skincells to help protect them against damage from sunlight (ultravioletlight). The amount of melanin in your skin is determined by geneticmakeup and one's exposure to sunlight. The more melanin pigment present,the darker the color of your skin.

Below the epidermis is the layer called the dermis. The top layer of thedermis, the one directly below the epidermis, has many ridges calledpapillae. On the fingertips, the skin's surface follows this pattern ofridges to create our individual fingerprints. The dermis contains avariable amount of fat, and also collagen and elastin fibres thatprovide strength and flexibility to the skin. In an older person theelastin fibres fragment and much of the skin's elastic quality is lost.This, along with the loss of subcutaneous fat, results in wrinkles.Blood vessels supply nutrients to the dividing cells in the basal layerand remove any waste products. They also help maintain body temperatureby dilating and carrying more blood when the body needs to lose heatfrom its surface; they narrow and carry less blood when the body needsto limit the amount of heat lost at its surface. The skin also containsa number of nerves and glands.

Overall skin quality and appearance can be affected by a variety ofdisorders, including aging, photoaging, acne, enlarged pores, andscarring. The intrinsic process of chronological aging results fromthinning of the epidermis and dermis and loss of elasticity. Thisprocess affects all layers of the face, including subcutaneous tissue,the musculofascial system, the superficial musculoaponeurotic system,and the facial skeleton. The result is bony resorption, atrophy ofsubcutaneous fat, attenuation of the musculofibrous system, andalterations of skin surface. The dermal-epidermal junction flattens,which results in loss of rete ridges and a thinner appearance to theepidermis. The dermis also becomes thin, with a decrease in elasticfibers, collagen production, vascularity, and ground substance. Thebiochemical alterations in collagen and elastin result in a dermis thatis more lax yet less elastic and resilient. Collectively, these changesresult in fine wrinkling of the skin and sagging of the tissues thatoverlay the facial skeleton.

In some embodiments, the invention relates to composition comprisingextracts that can stimulate skin cells to regenerate spontaneously. Inadditional embodiments, cells with elongated telomeres made in situ fromthe subject's own cells are reintroduced to the subject.

Many modalities can resurface the skin to improve skin quality, reduceage spots, soften fine lines, and treat acne or other scars. Modalitiesinclude traditional dermabrasion, chemical peeling, laser resurfacing,and microdermabrasion. The techniques attempt to remove the outer layersof skin with the idea that stimulating new growth will improveappearance. The initial evaluation of skin condition is typicallyaccomplished using Fitzpatrick's scale of sun-reactive skin types, whichdenotes patients' reactions to ultraviolet radiation and existing degreeof pigmentation. Type I patients always burn and never tan. Type IIpatients tan only with difficulty and usually burn. Type III patientstan but sometimes burn. Type IV patients rarely burn and tan with ease.Type V patients tan very easily and very rarely burn. Type VI patientstan very easily and never burn.

Chemical peeling is the chemical removal of layers of skin to improvedermatologic defects. The mechanism of action of peeling agents isrelatively straightforward. Stronger agents such as phenol (with variousadditives such as croton oil and glycerin) and trichloroacetic acid(TCA) produce a chemical necrosis of the skin to variable depths,depending on a number of controlled and uncontrolled variables. Theweaker agents change the pH sufficiently to cause a superficial shock tothe cells and, depending on many variables, cell injury or death. Whenused with a moisturizer, the acid acts simply to cause cellular andintercellular swelling and plumping, leading to transient increase incell and matrix size and lessening of fine lines and rhytides.Sequential treatments lead to exfoliation and a smoother complexion.Continued irritation can lead to many of the same effects of tretinoinor retinoid treatment (i.e., increased thickness of dermis, increasedblood flow to skin). The phenol peel “The Baker formula” is phenol USP88% 3 cm³ 49%; distilled water 2 cm³ 44%; croton oil 3 drops 2.1%; andSeptisol 8 drops 4.5%.

The microdermabrasion technique abrades the skin with a high-pressureflow of crystals. Microdermabrasion is most effective with superficialskin conditions because it produces a superficial depth of injury.Superficial skin conditions include early photoaging, fine lines, andsuperficial scarring. Microdermabrasion is accomplished by placing theskin under tension so that an effective vacuum is achieved. Typically,stretching the treatment area with the nondominant hand and using thedominant hand to guide the handpiece is the method used to achieve thiseffect. When treating the neck, the neck is placed in extension toassist in skin tension. The handpiece is moved over the treatment areain a single, smooth stroke, which can then be repeated. The pressure ofthe crystal stream is controlled with a foot pedal. Thicker skin, suchas that on the forehead, chin, and nose, can be treated moreaggressively (ie, adjust the speed of handpiece movement or number ofpasses). Decrease the pressure when treating the thinner skin of thelower eyelids and upper cheek. Vertically orient all strokes whentreating the neck.

Laser skin resurfacing (LSR) can be performed as an isolated procedureor as an adjunct to procedures such as transconjunctival blepharoplasty(TCB), facelift, and endoscopic browlift. The laser allows for precisecontrol of ablation depth, and it permits the surgeon to vary thesedepths as needed. In addition to such precision, LSR causes favorableheating of the dermis, which tightens collagen fibers and stimulatesneocollagen secretion by fibroblasts. Two laser wavelengths arepreferred for facial skin resurfacing: pulsed carbon dioxide anderbium:yttrium-aluminum-garnet (Er:YAG). Each Er:YAG pulse removes only25-30 micrometers of tissue compared to the pulsed carbon dioxide, whichremoves 50-100 micrometers. The Er:YAG produces less collateral dermalenergy because the thermal conduction is approximately 5 micrometers;pulsed carbon dioxide is 30-50 micrometers. The laser output of Er:YAGis directly absorbed by collagen and dermal proteins, whereas the carbondioxide laser vaporizes extracellular water in the dermis. Each Er:YAGpass generates the same amount of ablation, whereas the pulsed carbondioxide generates a decreased vaporization depth with each pass.

The composition of the present invention also finds use in woundhealing. A wound is a break in the skin (the outer layer of skin iscalled the epidermis). Wounds are usually caused by cuts or scrapes.Healing is a response to the injury that sets into motion a sequence ofevents. With the exception of bone, all tissues heal with some scarring.The object of proper care is to minimize the possibility of infectionand scarring.

Pressure ulcers are chronic wounds caused by unrelieved pressure thatresults in tissue damage. The ulcers are staged from I to IV, accordingto the level of tissue damage observed. Pressure ulcers are most commonin hospitalized patients, nursing home patients and those with spinalcord injuries. The standard of care for pressure ulcers includesinterval dressing changes, pressure relief, repositioning, physicalstrengthening, nutritional support and infection management. If thewound becomes severe, surgical interventions include wound debridementand skin-flap, muscle-flap or free-flap reconstruction.

The present invention also finds use for the treatment of various skindisorders. Uneven skin, discoloration, and growths can be caused by avariety of factors including genetics, exposure to sun, and/or use ofmedications. Callus formation (Clavus) is a thickening of the skin dueto intermittent pressure and frictional forces. The shape of the handsand feet are important in clavus formation. Specifically, the bonyprominences of the metacarpophalangeal and metatarsophalangeal jointsoften are shaped in such a way as to induce overlying skin friction. Asclavus formation ensues, friction against the footwear is likely toperpetuate hyperkeratosis. Toe deformity, including contractures andclaw, hammer, and mallet-shaped toes, may contribute to pathogenesis.Bunionettes, ie, callosities over the lateral fifth metatarsal head, maybe associated neuritic symptoms due to compression of the underlyinglateral digital nerves. Furthermore, Morton toe, in which the second toeis longer than the first toe, occurs in 25% of the population; this maybe one of the most important pathogenic factors in a callus of thecommon second metatarsal head, ie, an intractable plantar keratosis.

Moles (Nevi) are nests of melanocytes that are in contact with eachother. They typically start formation during early childhood. It hasbeen suggested that they form in response to sun exposure. However, agenetic factor is clearly involved in nevi. Some kinships express anautosomal dominant condition in which members have a large number oflarge nevi, sometimes more than 150 nevi scattered over the integument.Nevi have been observed to develop rapidly after blistering events, suchas second-degree thermal burns or sunburns; toxic epidermal necrolysis;and in persons with genetic blistering diseases, such as epidermolysisbullosa. Growth factors, such as basic fibroblast growth factor, havebeen suggested to be released by proliferation keratinocytes and tostimulate melanocyte proliferation. Melanocytic nevi are benignneoplasms or hamartomas composed of mostly melanocytes, thepigment-producing cells that colonize the epidermis. Melanocytes arederived from the neural crest and migrate during embryologic developmentto selected ectodermal sites (primarily the skin and the CNS) but alsoto the eyes and the ears. Ectopic melanocytes have been identified atautopsy in the gastrointestinal and genitourinary tracts. Congenitalmelanocytic nevi are thought to represent an anomaly in embryogenesisand as such could be considered a malformation or a hamartoma. Incontrast, most acquired melanocytic nevi are considered to be benignneoplastic proliferations.

Atypical moles/dysplastic nevi are acquired melanocytic lesions of theskin whose clinical and histologic definitions are still evolving.Atypical moles differ from common acquired melanocytic nevi in severalrespects, including diameter and lack of pigment uniformity

Birth marks (Capillary hemangiomas) are one of the most common benignorbital tumors of infancy. They are benign endothelial cell neoplasmsthat are typically absent at birth and characteristically have rapidgrowth in infancy with spontaneous involution later in life. This is incontrast to another known group of childhood vascular anomalies,vascular malformations. Vascular malformations, such as lymphangiomasand arteriovenous malformations, are present at birth and arecharacterized by very slow growth with persistence into adult life.

Striae distensae (Stretch marks) affect skin that is subjected tocontinuous and progressive stretching; increased stress is placed on theconnective tissue due to increased size of the various parts of thebody. It occurs on the abdomen and the breasts of pregnant women, on theshoulders of body builders, in adolescents undergoing their growthspurt, and in individuals who are overweight. Skin distension apparentlyleads to excessive mast cell degranulation with subsequent damage ofcollagen and elastin. Prolonged use of oral or topical corticosteroidsor Cushing syndrome (increased adrenal cortical activity) leads to thedevelopment of striae.

Acne manifestation is defined by the distribution of the pilosebaceousglands. Adolescence causes endocrine maturation of the adnexal elements,resulting in an accumulation of cellular products within the ductilesystems. In addition to the cellular products are coexistentmicroorganisms, most commonly Propionibacterium acnes and Staphylococcusepidermidis.

Rosacea is a common condition characterized by symptoms of facialflushing and a spectrum of clinical signs, including erythema,telangiectasia, coarseness of skin, and an inflammatory papulopustulareruption resembling acne. Rosacea is defined by persistent erythema ofthe central portion of the face lasting for at least 3 months.Supporting criteria include flushing, papules, pustules, andtelangiectasias on the convex surfaces. Secondary characteristics areburning and stinging, edema, plaques, a dry appearance, ocularmanifestations, and phymatous changes. Perioral dermatitis (POD) is achronic papulopustular facial dermatitis. It mostly occurs in youngwomen. The clinical and histologic features of the lesions resemblethose of rosacea.

Warts are benign proliferations of skin and mucosa caused by the humanpapilloma virus (HPV). Currently, more than 100 types of HPV have beenidentified. Certain HPV types tend to occur at particular anatomicsites; however, warts of any HPV type may occur at any site. The primaryclinical manifestations of HPV infection include common warts, genitalwarts, flat warts, and deep palmoplantar warts (myrmecia). Less commonmanifestations of HPV infection include focal epithelial hyperplasia(Heck disease), epidermodysplasia verruciformis, and plantar cysts.Warts are transmitted by direct or indirect contact, and predisposingfactors include disruption to the normal epithelial barrier. Treatmentcan be difficult, with frequent failures and recurrences.

Genital warts are a result of human papillomavirus (HPV) infectionacquired by inoculation of the virus into the epidermis via defects inthe epithelium (eg, maceration of the skin). Autoinoculation of virusinto opposed lesions is common. Spread of HPV infection is usuallythrough skin-associated virus and not from blood-borne infection.

Bowenoid papulosis (BP) occur on the genitalia of both sexes in sexuallyactive people. BP is manifested as papules that are induced virally byhuman papillomavirus (HPV) and demonstrate a distinctive histopathology(bowenoid dysplasia).

Psoriasis is characterized by exceedingly rapid turnover of skin andappears as a chronic, bilaterally symmetric, erythematous plaquelikelesion with a silvery scale covering. The lesions classically arelocated over the extensor surfaces, including the elbows, knees, back,and scalp. Confluent generalized lesions also may occur.

In Von Recklinghausen disease multiple neural tumors appear on the body.Numerous pigmented skin lesions occur. The classic café au lait spotspredominate. Additionally, pigmented iris hamartomas (i.e., Lischnodules) are common. Bone lesions and intracranial and GI lesions andsymptoms may be identified.

Necrobiosis lipoidica diabeticorum is a plaquelike, depressed, atrophicyellow lesion typically found in patients with diabetes. It has a strongassociation with diabetes and actually may be a clinical prodrome of theonset of the disease systemically. It rarely is found in locations otherthan the lower extremities and seldom is found in the absence ofdiabetes. The lesion tends to progress from a red plaquelike area to onewith atrophy that occasionally may ulcerate.

Seborrheic dermatitis is a papulosquamous disorder patterned on thesebum-rich areas of the scalp, face, and trunk. In addition to sebum,this dermatitis is linked to Malassezia, immunologic abnormalities, andactivation of complement.

Seborrheic keratosis (also known as seborrheic wart, senile wart, andbasal cell papilloma) is a common benign tumor in advanced andmiddle-aged persons. It is typically a raised papular lesion of variablecolor from light to dark brown. Seborrheic keratosis may be smooth orwartlike with visible pitting. Common sites include the face, trunk, andextremities. The lesion also may be pedunculated or sessile. A variantknown as dermatosis papulosa nigra occurs over the forehead and malarregions of individuals with black skin.

Acrochordons (also known as skin tag, fibroepithelial polyp, fibromamolle, and fibroepithelial papilloma) occasionally are associated withpregnancy, diabetes mellitus, and intestinal polyposis syndromes. Theytend to be located in the intertriginous areas of the axilla, groin, andinframammary regions as well as in the low cervical area along thecollar line. They are soft fleshy papules and usually, although notnecessarily, pedunculated.

Actinic keratosis is the most common sun-related growth. Actinickeratoses are chiefly found on the sun-exposed areas of the face, theears, the forearms, and the dorsum of the hands. However, they may occuron any area that is chronically or repeatedly exposed to the sun, suchas the back, the chest, and the legs. They usually appear as multiplediscrete, flat or elevated, verrucous, keratotic lesions. Lesionstypically have an erythematous base covered by scale (hyperkeratosis).They are usually 3-10 mm in diameter and gradually enlarge into broader,more elevated lesions. With time, actinic keratoses may develop intoinvasive cutaneous horns or skin cancers. Histologically, the epidermalchanges are characterized by acanthosis, parakeratosis, anddyskeratoses. Cellular atypia is present, and the keratinocytes vary insize and shape. Mitotic figures are common.

The compositions of the present invention also find use in the treatmentof burns. Sunburn is an acute cutaneous inflammatory reaction thatfollows excessive exposure of the skin to ultraviolet radiation (UVR).Exposure to solar radiation has the beneficial effects of stimulatingthe cutaneous synthesis of vitamin D and providing radiant warmth.Unfortunately, when the skin is subjected to excessive radiation in theultraviolet range (wavelength <400 nm), deleterious effects may occur.The most common is acute sunburn or solar erythema. Eyes, particularlythe cornea (the clear window of tissue on the front of the eyeball), canbe damaged easily by exposure to ultraviolet radiation from the sun andfrom other sources of ultraviolet light, such as a welder's arc, aphotographer's flood lamps, a sun lamp, or even a halogen desk lamp.

Epithelialization, angiogenesis, granulation tissue formation, andcollagen deposition are involved in the proliferation phase anabolicportion of wound healing. Epithelialization occurs early in woundrepair. If the basement membrane remains intact, the epithelial cellsmigrate upwards in the normal pattern. This is equivalent to afirst-degree skin burn. The epithelial progenitor cells remain intactbelow the wound, and the normal layers of epidermis are restored in 2-3days. If the basement membrane has been destroyed, similar to a second-or third-degree burn, then the wound is reepithelialized from the normalcells in the periphery and from the skin appendages, if intact (eg, hairfollicles, sweat glands)

Angiogenesis, stimulated by TNF-alpha, is marked by endothelial cellmigration and capillary formation. The new capillaries deliver nutrientsto the wound and help maintain the granulation tissue bed. The migrationof capillaries into the wound bed is critical for proper wound healing.The granulation phase and tissue deposition require nutrients suppliedby the capillaries, and failure for this to occur results in achronically unhealed wound. Mechanisms for modifying angiogenesis areunder study and have significant potential to improve the healingprocess.

During granulation tissue formation, fibroblasts differentiate andproduce ground substance and then collagen. The ground substance isdeposited into the wound bed; collagen is then deposited as the woundundergoes the final phase of repair. Many different cytokines areinvolved in the proliferative phase of wound repair. The steps and theexact mechanism of control are not well understood. Some of thecytokines include PDGF, insulinlike growth factor (IGF), and EGF.

During a remodeling stage, the framework (collagen) becomes moreorganized making the tissue stronger. The blood vessel density becomesless, and the wound begins to lose its pinkish color. Over the course of6 months, the area increases in strength, eventually reaching 70% of thestrength of uninjured skin. In the maturational phase, the woundundergoes contraction, ultimately resulting in a smaller amount ofapparent scar tissue. The entire process is a dynamic continuum with anoverlap of each phase and continued remodeling. The wound reachesmaximal strength at one year, with a tensile strength that is 30% ofnormal skin. Collagen deposition continues for a prolonged period, butthe net increase in collagen deposition plateaus after 21 days.

Epithelialization is the process of laying down new skin, or epithelial,cells. The skin forms a protective barrier between the outer environmentand the body. Its primary purpose is to protect against excessive waterloss and bacteria. Reconstruction of this layer begins within a fewhours of the injury and is complete within 24-48 hours in a clean,sutured (stitched) wound. Open wounds may take 7-10 days because theinflammatory process is prolonged, which contributes to scarring.Scarring occurs when the injury extends beyond the deep layer of theskin (into the dermis).

Collagen production is elevated in keloid biopsy samples and in culturedfibroblasts derived from keloids. Increased collagen production bycultured fibroblasts derived from keloids persists throughout their invitro life span; they do not revert to normal after transfer of thelesion to culture. No significant differences in DNA content orcellularity are found when keloid dermis is compared with normal dermis.This suggests that each fibroblast is producing more collagen ratherthan an increase occurring in the number of fibroblasts producing anormal amount of collagen. In keloid formation, excessive collagenproduction by fibroblasts is likely due to the wound environment.

Widened scar formation is thought to result from wound edge separationwith tension perpendicular to the healing skin wound. A state of tensionexists naturally in skin; wounded skin gapes and becomes ellipticalrather than round. When a wound is closed opposite to the lines oftension, the chance of widened scar formation is increased.

Upon clinical examination, keloids and hypertrophic scars are raisedabove the skin level. Hypertrophic scars are self-limited; theyhypertrophy within the confines of the wound. Initially, hypertrophiedscars can be raised, red, pruritic, and even painful; however, overtime, they become pale and flat. Hypertrophied scars appear worst at 2weeks to 2 months. Keloid scars can be differentiated from hypertrophicscars by their spread beyond the original wound. Keloid scars tend toremain red, pruritic, and painful for many months to years untilmenopause. Patients usually have a personal or familial history ofkeloid formation. Different from hypertrophic and keloid scars, widenedscars are flat and sometimes depressed. With adequate wound maturation,these wounds fade to the pigment of the surrounding uninjured skin.Widened scars are not usually red or pruritic.

The relaxed skin tension lines follow furrows formed when the skin isrelaxed. Unlike wrinkles, they are not visible features of the skin.They are merely derived from the furrows produced by pinching on theskin. These furrows are produced preferably with pinching perpendicularto the lines. When the skin is pinched oblique to the relaxed skintension lines, an S-shaped pattern is created. Fewer and higher furrowsare created if skin is pinched parallel to the lines. Closing incisionsopposite to the relaxed skin tension lines can increase the risk ofwidened or hypertrophic scar formation.

A potential relative contraindication to scar revision surgery existswhen the scar is a keloid because of the risk of worsening the scar.Sometimes, when keloids recur, they become larger than the original.Widened scars can be easily differentiated from hypertrophic and keloidscars based on findings from a physical examination. Widened scars areflat and sometimes even depressed. Hypertrophic scars and keloids areindistinguishable under light microscopy. However, there are a number ofdifferences when viewed under an electron microscope and when evaluatedimmunochemically. Keloids contain thick collagen fibers with increasedepidermal hyaluronic content, whereas hypertrophic scars exhibit nodularstructures with fine collagen fibers and increased levels of alphasmooth muscle actin. The collagen in both keloids and hypertrophic scarsis organized in discrete nodules, frequently obliterating the rete pegsin the papillary dermis of the lesions. While collagen in normal dermisis arranged in discrete fascicles separated by considerable interstitialspace, collagen nodules in keloids and in hypertrophic scars appearavascular and unidirectional and are aligned in a highly stressedconfiguration.

Different nonsurgical options treat abnormal scars. Pressure is thoughtto decrease tissue metabolism and increase collagen breakdown within thewound. The different methods of applying pressure include the use ofelastic bandages (ACE wraps), thromboembolic disease stockings, orIsotoner-type gloves on extremities. Alternatively, custom-fittedcompression garments can be used to apply pressure to the more difficultareas, including the neck and torso. Because these devices areuncomfortable, patient compliance varies. Unfortunately, for optimalresults, these devices must be used for 6-12 months during thematuration of the wound.

Silicone gel can be used to treat abnormal scars. Silicone gel is shownto significantly decrease scar volume when used over time particularlyfor hypertrophic scar formation. The effect of the silicone gel on thescar is believed to be due to wound hydration. The silicone gel isapplied to the wound for at least 12 h/d. Patients find it moreappealing to apply the silicone to their wounds at night. Silicone gelis gaining popularity because it can be applied to a smaller area for 12h/d, usually at night. However, skin breakdown, rashes, and difficultywith wound adherence can lead to disuse.

Steroid injections have become a common nonsurgical option in thetreatment of problem scars. The steroid used for intralesional injectionis triamcinolone (Kenalog). Triamcinolone injections have been thestandard treatment to induce flattening, fading, and decreasedsymptomatology of hypertrophied scars. These injections can beadministered as soon as a problem scar is identified. The dose of theinjection can vary from 10-120 mg, depending on the size of the scar.

One may make use of a triamcinolone injection for thin-to-widehypertrophied scars and silicone for very wide hypertrophied scars. Somepatients prefer triamcinolone injections to avoid applying and wearingthe silicone every day for 6-9 months, especially on body areas whereadherence is poor. Adverse effects of triamcinolone injections includehypopigmentation and subcutaneous atrophy. Other nonsurgical optionsinclude corticosteroid intralesional injections, vitamin E therapy, zincoxide therapy, antineoplastic agents, and immunotherapy.

If nonoperative measures are unsuccessful in the treatment of abnormalscars, operative intervention can be considered. Closing wounds toorient the wound along the relaxed skin tension lines is important. Astandard practice often used rather subconsciously after excision of alesion involves assessing the direction of least tension based on theconfiguration of the edges of the wound or by pinching the wound.

The first-line procedure used for scar revision is fusiform excision. Ingeneral, fusiform excision does not require lengthening the scar. Inorder to avoid canine auricles, ensure the wound has a length-to-widthratio of 4:1. Fusiform excision is preferred for short wounds orientedalong relaxed skin tension lines. The Millard flap procedure is similarto fusiform excision, but it involves preserving the scar and itsconnection to the underlying fat. The skin is incised in a fusiformfashion around the scar to the subcutaneous level. The scar is thendeepithelialized, and the skin edges are approximated over thedeepithelialized scar. The Millard flap technique is preferred forwidened, depressed scars.

Scars not oriented along the relaxed skin tension lines can be modifiedwith a Z-plasty procedure. Limbs of equal length are created for the Zplasty. The angle of the Z dictates the length of scar tensiondistribution and elongation (eg, 30° for 25%, 45° for 50%, 60° for 75%,75° for 100%, 90° for 120%). The W-plasty technique for scar revision issimilar to Z plasty because of the result of breaking up a straight-linescar into a pattern that is less conspicuous. Similar to a fusiformexcision, W plasty involves the removal of skin; therefore, avoid thismethod if significant tension is present across the wound edges.W-plasty scar revision is preferred for scars along relaxed skin tensionlines; scars with a bowstring contracture; short, depressed scars; andfacial scars.

Tissue expansion and serial excision can be considered for larger scarrevisions when excess wound tension is predicted. If more than 2 serialexcisions are expected, tissue expansion is preferred. Finally, otherprocedures that have been described to treat scars include dermabrasion,cryosurgery, and laser therapy. Widened scars may be treated differentlythan hypertrophied scars. Widened scars can be flat or even depressed.Therefore, the administration of intralesional steroids is notpreferred; these agents might worsen the depression. Widened scars arepreferably treated with the Millard 2-flap technique over adeepithelialized scar. This technique provides soft tissue fill underthe approximated wound edges. Furthermore, if the widened scar recurs,the risk for another recurrence may be minimized by reorienting thewound tension along the lines of relaxed skin tension. Other adjunctsdescribed in the treatment of widened scars include the injection of fatgrafts or other tissue substitutes. When oriented close to the relaxedskin tension lines, hypertrophic scars can be excised in a fusiformfashion. If the hypertrophic scar developed because of excessive tensionacross the wound as a result of unfavorable wound orientation, Z plastycan sometimes help reorient the wound to distribute tension in adifferent direction to minimize the risk of recurrence.

Postoperatively, compression garments and silicone gel are preferred for4-6 months to decrease the risk of recurrence. Patients are encouragedto refrain from strenuous activities for at least 6 weeks, until whichtime the wound achieves approximately 80% original wound tensilestrength. Patients are monitored for 6 months postoperatively to detectand potentially circumvent recurrences early. Postoperatively, patientsare at risk for hypertrophic scar and widened scar recurrence. Otherrisks include infection, hematoma, seroma, and painful or unattractivescarring. The risk of recurrence is significant for both hypertrophicand widened scars, and it is increased with repeat operations. Woundhealing requires approximately 1 year, during which time the surgeon andpatient should observe for and expect improvement. Once the scar has hadan opportunity to mature, scar revision can be considered.

In some embodiments of the present invention, compositions comprisingcell extracts are utilized to improve any area of the person visible andcontributing to cosmetic appearance of a person, including but notlimited to skin, hair, nails, teeth, subcutaneous fat, cartilage, muscleand skeletal structures. The described gene-gun and microinjectiondelivery methods are contemplated to introduce extracts or extractcomponents to structures below the surface skin of a person.

This invention relates to prevention of deterioration, damage andmalfunction of cells and tissues, and to promote, improve and exceedcellular function in order to promote, improve and exceed appearance,vitality and health by treating cells and tissues with differentiablecells, cell or egg extracts, or components of said extracts includingsignaling molecules, peptides, carbohydrates, lipids or nucleic acids.

The current invention contemplates the assessment of a persons needs forhealing, regeneration or repair of damage by several means, includingbut not limited to analysis and measurements of visible surfaces, skinpH, thickness, structure and elasticity of skin layers, analysis ofblood or tissue samples by microchip, RT-PCR, Mass spectrometry, highpressure liquid chromatography, ELISA-assays, RNA analysis, analysis ofaccumulation of DNA damage or defective genes by DNA sequencing,assessment of internal organ and tissue health by X-ray imaging,ultrasound imaging, computed tomogpraphy (CT), magnetic resonanceimaging (MRI), positron emission tomography (PET).

Subcutaneous fat contributes to the cosmetic appearance of a person, andis redistributed during ageing, by smoking and in a number of diseases,including HIV and diabetes as well as in burn-victims. The humanimmunodeficiency virus (HIV)-lipodystrophy syndrome is associated withfat redistribution and metabolic abnormalities, including insulinresistance. Increased intramyocellular lipid (IMCL) concentrations arethought to contribute to insulin resistance, being linked to metabolicand body composition variables. Among HIV-infected subjects, calfsubcutaneous fat area and extremity fat are reduced. Extremity fat issignificantly associated with IMCL among HIV-infected patients,controlling for visceral abdominal fat, abdominal subcutaneous fat, andantiretroviral medications in a regression model. Increased IMCL inHIV-infected women with a mixed lipodystrophy pattern are mostsignificantly associated with reduced extremity fat. (Torriani M et al.,J Appl Physiol. 2006 February; 100(2):609-14. Epub 2005 Oct. 13).Saturation of the subcutaneous fat depot is the primary event in thepathophysiology of insulin resistance in the majority of patients andpostulate that this seminal event may lead to the development ofhypertension, hypertriglyceridemia and depressed HDL levels (i.e., themetabolic syndrome). There are no current effective means toredistribute subcutaneous fat in such persons, current treatment include(1) weight loss with differing responses seen with regards to insulinresistance depending on the size of the fat depot; (2) peroxisomeproliferator activated receptor gamma agonists, such asthiazoledinediones which expand the subcutaneous fat depot, (3)expanding alternate storage sites for triglycerides by a variety oftechniques, such as resistance training-induced muscle hypertrophy, mayalso improve insulin resistance; (4) drugs, such as beta 3 adrenergicreceptor agonists which promote lipolysis may increase insulinresistance by releasing free fatty acids into the circulation.Inhibitors of the beta oxidation of free fatty acids (e.g., carnitinepalmitoyl transferase inhibitors) may cause insulin resistance bysparing fat and (5) liposuction, by reducing the size of thesubcutaneous fat depot may worsen insulin resistance, thus increasingthe risk of type 2 diabetes mellitus (Cherian M A, Santoro T J, MedHypotheses. 2005 Dec. 14; [Epub ahead of print]).

Alterations in subcutaneous fat and skin condition due to hormonechanges that occur during ageing and disease are also contemplated areasof use for this invention. Effects of ovarian and other steroids areimportant to the metabolism of skin and hair, the changes in bodycomposition and the alterations of the subcutaneous fat distributionthroughout life. So called aesthetic endocrinology accesses deficiencyor excess of ovarian steroids that lead to different problems skin andhair and other non-genital, i.e., obesity and cellulite. Sex steroidsare small molecules that are transported into the skin by topicalapplication when properly formulated, and are contemplated to be addedto the extracts presented in this invention in order to achieve localeffects but to avoid systemic reactions. Estrogens, delivered orally ortopically, may counteract the aging of the skin especiallypost-menopause. Estrogen alone is not sufficient for reconstitution ofjuvenile skin but may slow the skin aging process. The hitherto onlysuccessful treatment of hair loss in women is by application of thenon-hormonal compound minoxidil, and compositions contemplated by thisinvention may serve to be a different way of treating hair loss. Indeed,the compositions contemplated may stimulate hair sack follicles toregrow or increase the rate and quality of hair, as well as nails.Estrogens also contribute to hirsutism (the excessive growth of thickdark hair in locations where hair growth in women usually is minimal orabsent), acne and changes in body composition. (Gruber C J, et al.,Current concepts in aesthetic endocrinology. Gynecol Endocrinol. 2002December; 16(6):431-41). The compositions in the present invention areadditionally contemplated for use in hair loss and baldness in maleswhich may be caused by hormones, diet, cancer, chronic illness orstress.

It is contemplated that the present invention can be used to regulatehair growth by stimulating or modulating hair follicle cells to eitherreduce or enhance or regenerate hair growth in desired areas by topicalor sub-dermal applications.

This invention is also useful in the treatment of cellulite. Celluliteis a common term used to describe superficial pockets of trapped fat,which cause uneven dimpling or “orange peel” skin. It appears in 90% ofpost-adolescent women and is rarely seen in men. Common but notexclusive areas where cellulite is found, are the thighs, buttocks, andthe abdomen. Contrary to popular belief, cellulite is not related toobesity, since it occurs in overweight, normal, and thin women.Cellulite can be aided by mechanized devices with motorized rollers andregulated suction. This non-surgical and non-invasive device creates asymmetrical skin fold, which allows for deep tissue mobilization tooccur and results in reduction of cellulite and loss of inches. Thepresent invention contemplates application of extracts topically orsubcutaneously to regulate the distribution of subcutaneous fat depositsand improve the cosmetic appearance of areas affected by cellulite.

It is contemplated that the present invention may be useful for therepair or rejuvenation or de novo formation of damaged tissues, organsand cells beneath the skin, including all internal organs and tissues,including but not limited to muscle, fat, cartilage, bone, connectivetissue, spleen, liver, pancreas, lungs and nervous tissue. Damages tothe internal tissues or organs may be induced by i.e. accidents,diseases, medication, cancer, radiation and surgery.

When the body is exposed to high doses of radiation, a complexbiological response is initiated that may lead to multi-organ failure(MOF). MOF begins with energy deposits in cellular targets and ispropagated and amplified by the tissue response to cell damage. Thebiology of wound healing is at the root of MOF following surgicaltrauma, inflammation is the basis for MOF in sepsis, and the biology ofthe irradiated tissue initiates radiogenic MOF. Tissue response toradiation damage has been suggested to be initiated and co-ordinated byextracellular signaling. It has been demonstrated that transforminggrowth factor-β1 orchestrates the biology of irradiated tissue as atissue level sensor of oxidative stress, and is integral to the cellularDNA damage response.

(Barcellos-Hoff M H. How tissues respond to damage at the cellularlevel: orchestration by transforming growth factor-β (TGF-β) BritishJournal of Radiology (2005) Supplement_(—)27, 123-127).

In some embodiments, the compositions described above are used toincrease collagen production by skin cells. In some embodiments, thecompositions are applied to the skin or wounds in the skin in aneffective amount, which is the amount required to increase collagenproduction in the cells. It is contemplated that by increasing collagenproduction, the compositions of the present invention enhance or improvewound healing in a subject. It is also contemplated that by increasingcollagen production upon topical application, the compositions of thepresent invention can improve attributes of damaged skin, such asgeneral appearance, suppleness, smoothness, amount of wrinkles,moisture, color, etc. Accordingly, the composition of the presentinvention find use in increasing the collagen content in skin that hasbeen contacted by the composition so that skin moisture is improved orincreased, skin wrinkling is improved or decreased, skin suppleness isimproved or increased, skin smoothness is improved or increased, skintone is improved or increased, skin color is improved or normalized,skin stretch marks are improved, decreased, or eliminated or skinroughness is improved or decreased. In other embodiments, thecompositions of the present invention are useful for the prophylaxis orprevention of the foregoing skin conditions.

In some embodiments, the compositions described above are used toincrease the proliferation of skin cells, and in particular skinfibroblasts. In some embodiments, the compositions are applied to theskin or wounds in the skin in an effective amount, which is the amountrequired to increase fibroblast proliferation at the site ofapplication. It is contemplated that by increasing fibroblastproliferation, the compositions of the present invention enhance orimprove wound healing in a subject. It is also contemplated that byincreasing fibroblast proliferation upon topical application, thecompositions of the present invention can improve attributes of damagedskin, such as general appearance, suppleness, smoothness, amount ofwrinkles, moisture, color, etc. Accordingly, the composition of thepresent invention find use in increasing the collagen content in skinthat has been contacted by the composition so that skin moisture isimproved or increased, skin wrinkling is improved or decreased, skinsuppleness is improved or increased, skin smoothness is improved orincreased, skin tone is improved or increased, skin color is improved ornormalized, skin stretch marks are improved, decreased, or eliminated orskin roughness is improved or decreased.

EXAMPLES Example 1 Cells and Cell Extracts

NCCIT, Jurkat (clone E6-1) and 293T cells (American Type CultureCollection, Bethesda, Md.) are cultured in RPMI 1640 (Sigma, St. Louis,Mo.) with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodiumpyruvate and non-essential amino acids (complete RPMI). NIH3T3Swiss-Albino fibroblasts (American Type Culture Collection) are culturedin Dulbecco's modified Eagle's medium (DMEM; Sigma) with 10% FCS,L-glutamine and 0.1 mM β-mercaptoethanol. Mouse ESCs are isolated frominner cell masses of strain sv129 blastocysts and plated on mousefibroblast γ-irradiated feeder layers in ESC medium (DMEM, 15% FCS, 0.1mM β-mercaptoethanol, non-essential amino acids, 1%penicillin/streptomycin) supplemented with 1,000 units/ml (10 ng/ml) ofrecombinant leukemia inhibitory factor (LIF; Sigma) on gelatin-coatedplates. Prior to harvesting for preparing extracts, ESCs are passagedand cultured under feeder-free conditions in RPMI containing 10 ng/mlLIF.

To prepare NCCIT extracts, cells are washed in phosphate buffered saline(PBS) and in cell lysis buffer (100 mM HEPES, pH 8.2, 50 mM NaCl, 5 mMMgCl₂, 1 mM dithiothreitol and protease inhibitors), sedimented at 400g, resuspended in 1 volume of cold cell lysis buffer and incubated for30-45 min on ice. Cells are sonicated on ice in 200-μl aliquots using aLabsonic-M pulse sonicator fitted with a 3-mm diameter probe (B. BraunBiotech, Melsungen, Germany) until all cells and nuclei are lysed, asjudged by microscopy. The lysate is sedimented at 15,000 g for 15 min at4° C. to pellet the coarse material. The supernatant is aliquoted,frozen in liquid nitrogen and stored. Lysate of 95,583±10,966 NCCITcells is used to generate extract. ESC extracts (25-30 mg/ml protein)are similarly prepared from LIF-adapted ESC cultures. 293T, Jurkat andNIH3T3 extracts are also prepared as above. If necessary, extracts arediluted with H₂O prior to use to adjust osmolarity to ˜300 mOsm.

Example 2 Bulge Hair-Follicle Stem Cells

To isolate the vibrissa follicles, the upper lip containing the vibrissapad of a subject is cut and its inner surface was exposed. In humanindividuals, hairs from the scalp or other haired body parts may be usedinstead of vibrissa. The vibrissa or hair follicles are dissected undera binocular microscope. The vibrissa are plucked from the pad by pullingthem gently by the neck with fine forceps. The isolated vibrissae werewashed in DMEM-F12 (GIBCO/BRL), containing B-27 (GIBCO/BRL) and 1%penicillin/streptomycin (GIBCO/BRL). All surgical procedures were doneunder a sterile environment. The vibrissa follicular bulge areacontained nestin expressing cells. The cells were isolated by exposureto fluorescent anti-nestin antibodies under fluorescence microscopy. Theisolated cells were suspended in 1 ml of DMEM-F12 containing B-27 with1% methylcellulose (Sigma-Aldrich), and 20 ng·ml⁻¹ basic FGF (bFGF)(Chemicon). Cells were cultured in 24-well tissue-culture dishes(Corning) at 37° C. in a 5% CO₂/95% air tissue-culture incubator. After4 weeks, the bulge-area cells form colonies.

Example 3 Ex Vivo Therapy

Cells to be reprogrammed ex vivo are washed in cold PBS and in coldCa2+- and Mg2+-free Hank's balanced salt solution (HBSS; Invitrogen,Gaithersburg, Md.). Cells are resuspended in aliquots of 100,000cells/100 μl HBSS, or multiples thereof, placed in 1.5 ml tubes andcentrifuged at 120 g for 5 min at 4° C. in a swing-out rotor. Sedimentedcells are suspended in 97.7 ml cold HBSS, tubes placed in a H₂O bath at37° C. for 2 min and 2.3 ml SLO (Sigma; 100 mg/ml stock diluted 1:10 incold HBSS) is added to a final SLO concentration of 230 ng/ml. Samplesare incubated horizontally in a H₂O bath for 50 min at 37° C. withoccasional agitation and set on ice. Samples are diluted with 200 mlcold HBSS and cells are sedimented at 120 g for 5 min at 4° C.Permeabilization is assessed by monitoring uptake of a 70,000 Mr Texasred-conjugated dextran (Molecular Probes, Eugene, Oreg.; 50 μg/ml) in aseparate sample 24 h after resealing and replating the cells.Permeabilization efficiency under these conditions is ˜80%.

Following permeabilization, cells to be reprogrammed ex vivo aresuspended at 1,000 cells/μl in 100 ml extract (or multiples thereof)containing an ATP-regenerating system (1 mM ATP, 10 mM creatinephosphate, 25 mg/ml creatine kinase; Sigma), 100 μM GTP (Sigma) and 1 mMof each nucleotide triphosphate (NTP; Roche Diagnostics, Mannheim,Germany). The tube containing cells is incubated horizontally for 1 h at37° C. in a H₂O bath with occasional agitation. To reseal plasmamembranes, the extract is diluted with complete RPMI containing 2 mMCaCl₂ and antibiotics, and cells are seeded at 100,000 cells per well ofa 48-well plate. After 2 h, floating cells are removed and plated cellsare cultured in complete RPMI. The reprogrammed cells can betransplanted back into patient.

Example 4 Cream Base for Use with Cell Extracts

Water—78%

Proteins—10%

-   -   e.g., Keratin, Filagrin, and/or Growth factors in trace amounts        (μM-mM amounts of EGF, IGF, IGFII, Insulin, Substance P,        Defensins, NGF)

Lipids—10%

-   -   Squaline 9%, Aliphaic waxes 12%, Sterol esters 33%, Diol esters        7%, Triglycerides 26%, Free sterols 9%, Other lipids 4%.

Cell extract or egg extract or components of extracts—2%

A cream base made from any combination of lipids and/or proteins and/orwater containing cell extracts.

Example 5 Preparation of Fish Egg Extracts

Fresh, unfertilized salmon (Salmo salar) eggs harvested from females inreproductive phase (late fall) are kept on ice, and the extractpreferably made immediately. It is possible to freeze dry eggs in acryoprotectant (e.g., 1.5 M 1,2-propanediol and 0.2 M sucrose) withoutdisrupting the egg membrane. Freezing should be gradual (−1° C./min) to−80° C. Eggs should be thawed and kept on ice throughout the extractpreparation procedure.

Eggs are washed twice in HBSS or seawater with protease inhibitors (10ug/ml). The washing solution is removed and the eggs are lysed andhomogenized in a pre-chilled Dounce glass-glass homogenizator. Thelysate is transferred to Beckman Ultra Clear polyallomer centrifugetubes (5 ml) while avoiding transfer of egg shells, and centrifugatedfor 15 min at 15.000 g at 4° C. in a Beckman ultracentrifuge usingSW55T1 rotor. Three fractions are thereby obtained; lipid top fraction,cytoplasmic middle fraction, and a bottom fraction containing eggshellsand nucleic debris. The cytoplasmic middle fraction is the collectedextract. This extract is expected to contain most cytosolic organellesincluding mitochondria, lysosomes and peroxisomes, should be clear andviscous, and have an orange tint. Protease inhibitors (10 ug/ml stock)are added and extracts are kept at −80° C.

Further fractionation of the cytoplasmic extract is possible.Centrifugation at 100,000 g at 4° C. for 60 minutes yields 2-3fractions, where the top/middle cytoplasmic fraction contains thecytosol with endoplasmic reticulum, SV and microsomes. The extract pH ismeasured by litmus paper, protein concentration measured by Bradfordassay, and osmolarity measured by osmometer.

Mid-blastula Zebra fish embryos are collected, liquid removed and frozento −20° C. To prepare the extract, embryos are thawed on ice, lysed andhomogenized by Dounce glass-glass homogenizator in a small amount ofeither HBSS or seawater (preferably less than 50% liquid v/v). Thelysate is filtered through a sterile linen cloth and centrifugated at5,000 g at 4° C. for 20 minutes in a SX4250 rotor using a Beckman X-22Rcentrifuge. The cytoplasmic extract (supernatant) is collected andprotease inhibitors (10 ug/ml) are added. The extract may be Milliporefiltered (0.22 um MilliQ sterile filter). The extracts are kept at −80°C. The extract pH is measured by litmus paper, protein concentrationmeasured by Bradford assay, and osmolarity measured by osmometer.

This general procedure is useful for the preparation of extracts fromsea urchin, shrimp, fish eggs/roe or frog eggs. Briefly, roe collectedfrom gravid female fish soon after they liberated their eggs in aspawning program (hCG hormone injected (1 ml/kg) at 6 to 8 hours beforeegg liberation, usually at dawn (2-4 am), or from gravid frogs. Roe/eggsare freeze dried or frozen at −20° C. or used fresh. Roe is collectedfrom different kinds of fish. For sea-urchin, 0.5 M KCl is injectedaround the mouth to evoke shedding of eggs. The extract is prepared fromeggs/roe by crushing (cell cracker or dounce-homogenization) orcentrifugation at different speeds to separate cytoplasm with allcontent, with/without egg-shells (zona pellucida), with/withoutnucleus/cytosol, with/without organelles, with/without lipids. Furtherfractionation can be conducted to isolate one or more of mRNA, proteins,small peptides, carbohydrates and lipids. Major components of fattyacids in the roe are oleic acid, linoleic acid, and omega-3 fatty acids.

Upon application of the above protocol for salmon egg extracts, thesalmon egg extracts had a surprisingly high protein concentrationvarying from 100-380 mg/ml, pH between 6.4-6.8, and an osmolarity ofapproximately 350 mOsm. The extracts were clear and viscous andnon-filterable (by 0.45 um MilliQ filter). The protein in the extractprecipitated easily upon addition of water or hydrous solutions with lowbuffering capacity due to the high protein content and low pH. Extractscould be neutralized to pH 7.0 by addition of alkaline (1-3 ul 1MNaOH/ml extract), whereupon dilution in water and hydrous solutions waspossible. Zebra-fish extracts had a protein concentration varying from23-26 mg/ml, pH between 6.4-6.8, and an osmolarity between 80-150 mOsm.The extracts were clear and non-viscous, filterable and diluted readilyin water at all dilutions.

Example 6 Toxicity Testing of Extracts

Extracts with low pH and that contain certain substances may be toxic tocells. Toxicity of each batch should be tested on each cell type that isto be reprogrammed Cells are harvested and washed twice in HBSS.Approximately 100,000 cells are pelleted and resuspended in 100 ulextract and incubated in a waterbath at 37° C. for 1 hour. Dilutions ofthe extracts may be tested to assess cell survival in extracts ofvarying protein concentration, pH and osmolarity. Optimally, proteinconcentration should be more than 25 mg/ml, pH should be close to 7.2,and osmolarity close to 280 mOsm. Cells and extract are incubated inwells with normal media (as suited to cell type chosen) for 24 hours,and the morphology of the cells inspected by microscopy. Cells areharvested, stained, and viable cells counted. If more than 50% of cellsare non-viable after culture, the extract is considered toxic.

Upon application of the above protocol, 293T cells were viable for atleast 3 weeks after incubation with extracts of salmon eggs and zebrafish embryo with protein concentrations varying from 24-380 mg/ml, atosmolarities between 140-350 and pH 6.9-7.7. At osmolarity below 140mOsm, the cells died.

Cellular morphology of cells reprogrammed with salmon egg extracts orextracts of zebrafish embryos changed after approx. 3 days. 293T cellsbecome rounder, and some populations of cells start to grow in blastomalike spheres. These changes are persistent, and can be observed until 21days (experiment terminated), although in certain conditions the changesseem to reverse towards normal 293T morphology after 2 weeks. Uponculture of normal 293T cells with extract added to normal media(RPMI-1640 with 10% FCS and 0.2% extract), similar changes in morphologycan be observed as seen for reprogrammed cells cultured in normal media.Additionally, cells cultured with salmon egg extracts in particular havean increased growth rate compared to normal cells. When starving cells(RPMI-1640 with 0.5% FCS), growth rate decreases significantly fornon-extract treated cells, and morphology of cells changes slightly. Forstarved cells grown with extracts (0.2% extract in starvation media),the changes are more pronounced. In this case, most cell populationsgrow in blastomer like spheres, and the spheres detach from the culturevessel and float in the media, where they keep growing. Interestingly,the deceleration in growth rate is reversed in cells cultured withextract added to the starvation medium.

Example 7 Physical Properties of Extracts

RNA, DNA and protein content of LEX were measured using the Qube-iTfluorimeter from InVitrogen. All extracts measured have yieldedcomparable effects on collagen secretion from human fibroblasts in vitroat 0.5% stimulation for 8 days. Extracts were diluted in PBS and Qube-iTassay buffer prior to measurements.

RNA Content of Salmon and Trout Homogenates and Extracts Average 2-5mg/ml.

Homogenates of salmon eggs (non-centrifugated) contain 3-4 mg/ml RNAAfter centrifugation to 9-15,000 g, RNA content was reduced to 2-3mg/ml. This is probably due to RNA being centrifugated down or degraded.Interestingly, trout egg homogenates (non-centrifugated) contain 2-3mg/ml RNA, but after centrifugation to 9-15,000 g, the concentration ofRNA sis increased to 3-5 mg/ml. Extracts made from trout eggs are lessviscous than extracts made from salmon eggs, and may keep RNA better inwater phase suspension during centrifugation.

DNA Content of Salmon and Trout Homogenates and Extracts Between 40-500ug/ml.

Homogenates of salmon eggs (non-centrifugated) contain 60-200 ug/ml DNAAfter centrifugation to 9-15,000 g, DNA content was reduced to 40-51ug/ml. This is probably due to DNA being centrifugated down.Interestingly, homogenates of trout eggs (non-centrifugated) containmore DNA than salmon egg extracts: 130-530 ug/ml DNA. Aftercentrifugation to 9-15,000 g, DNA content is reduced to 70-125 ug/ml,but is still higher than comparable salmon egg extracts. Extracts madefrom trout eggs are less viscous than extracts made from salmon eggs,and may keep DNA in better water phase suspension during centrifugation.

The DNA content varies widely between test-homogenates prepared here,and may be caused by differential lysing of nuclei containing gDNA priorto centrifugation. Better lysing of nuclei by variations on thehomogenization process during production may yield extracts with higherDNA content. These differential extracts may yield separate effectsuseful for different applications, such as effects on gene expression inskin cells.

Protein Content of Salmon and Trout Homogenates and Extracts Average180-300 mg/ml.

Homogenates of salmon eggs (non-centrifugated) contain 180-260 mg/mlprotein. After centrifugation to 9-15,000 g, protein content wasunchanged or increased slightly to 200-260 mg/ml. Homogenates of trouteggs (non-centrifugated) contain 250-300 mg/ml protein, and aftercentrifugation to 9-15,000 g, protein content is roughly the same(250-270 mg/ml). The protein fraction of the egg cytosol is not expectedto be spun down at the g-forces applied, and may be expected to besimilar to the raw protein content of the egg cytosol.

Previous measurements of protein contents in extracts using a Nano-dropspectrophometer showed a range of 150-250 mg/ml. This may be due to anupper detection limit around 250 mg/ml in the Nano-drop. It is probablethat the slightly higher fluorometer measurements presented here aremore accurate.

TABLE 1 Summary of measurements RNA, DNA and protein content in extractsSource Centrifugation LEX/corresp mg/ml μg/ml mg/ml of eggs speed to LEXRNA DNA protein Salmon Homogenate, LEX20 3.51 66.8 256 no centrifugationSalmon 15000 × g LEX20 2.34 44 252 Salmon Homogenate, LEX24 3.42 192.4180 no centrifugation Salmon 12000 × g LEX24 2.93 50.8 208 TroutHomogenate, LEX28 2.67 131.6 249 no centrifugation Trout 15000 × g LEX283.51 73.2 249 Trout Homogenate, LEX25 2.53 528 296 no centrifugationTrout 15000 × g LEX25 3.70 72.8 262 Trout 15000 × g LEX25 3.63 99.2 210Trout 12000 × g LEX31 4.59 87.2 270 Trout 12000 × g LEX32 4.68 124.8 —Trout 12000 × g LEX33 4.67 94.4 252

Lipid Content of Extracts is 3.7-4.5 g/100 g Extract (3.7-4.5%).

The lipid content of extracts were measured by ALS (Germany), and wasfound to be in the narrow range of 3.7-4.5 g/100 g in all extracts fromsalmon or trout roe prepared at centrifugations spanning from 1,700 g to15,000 g. The lower g-force centrifugations appear to require spinningat room temperature to give equal lipid fractionation to higher g-forcesat 4 degrees centigrade. At lower centrifugal forces than 1,700 g alsoapplicable to produce an extract the lipid content may be higher (4-7%).The extract may contain the following lipids (right column), and thelipid fraction removed from the extract during production may includethe following lipids (left column)

TABLE 2 Lipid content can vary beetween batch production and specificlipids can include: Removed % of from remaining extract lipids in Reportby ALS Scandinavia LIPID extract ELEMENT SAMPLE lipid fract. LEX 42Fatty acids, saturated g/100 g 22.7 1.6 Fatty acids, monounsaturatedg/100 g 36.6 2.8 Fatty acids, polyunsaturated g/100 g 44.5 0.8 C4:0Butyric acid g/100 g <0.10 <0.10 C6:0 Caproic acid g/100 g <0.10 <0.10C8:0 Caprylic acid g/100 g <0.10 0.25 C10:0 Capric acid g/100 g <0.100.62 C11:0 Undecanoic acid g/100 g <0.10 <0.10 C12:0 Lauric acid g/100 g<0.10 <0.10 C13:0 Tridecanoic acid g/100 g <0.10 <0.10 C14:0 Myristicacid g/100 g 3 4.2 C14:1 Myristoleic acid g/100 g 0.11 0.15 C15:0Pentadecanoic acid g/100 g 0.3 0.34 C15:1 cis10-Pentadecanoic acid g/100g <0.10 0.12 C16:0 Palmitic acid g/100 g 11 14.3 C16:1 Palmitoleic acidg/100 g 7.3 9.6 C17:0 Heptadecanoic acid g/100 g 0.24 0.21 C17:1Heptadecenoic acid g/100 g <0.10 <0.10 C18:0 Stearic acid g/100 g 3.65.4 C18:1 Oleic acid g/100 g 22.9 33.8 C18:2 Linoleic acid (omega6)g/100 g 6.2 5 C18:3 Linolenic acid(omega6) g/100 g 0.92 0.47 C18:3a-Linolenic acid(omega3) g/100 g 2.4 <0.10 C18:4 Stearidonic acid (ome3)g/100 g 0.57 0.23 C20:0 Arachidic acid g/100 g 0.85 0.43 C20:1Eicosenoic acid g/100 g 0.83 1.1 C20:2 Eicosadienoic acid (om6) g/100 g0.49 0.34 C20:3 Eicosatrienoic ac(omega6) g/100 g 0.3 0.14 C20:4Arachidonic acid(omega6) g/100 g 1.6 0.78 C20:5 Eicosapentaenoicac(ome3) g/100 g 9.4 2.8 C21:0 Heneicosanoic acid g/100 g <0.10 <0.10C22:0 Behenic acid g/100 g 3.1 1.3 C22:1 Erucic acid g/100 g <0.10 <0.10C22:2 Docosadienoic ac (ome6) g/100 g <0.10 <0.10 C22:5 Docosapentaenoicac(ome3 g/100 g 6.8 2.3 C22:6 Docosahexaenoic ac(ome3) g/100 g 13.5<0.10 C24:0 Lignoceric acid g/100 g <0.10 <0.10 C24:1 Nervonic acidg/100 g <0.10 2.6 Vanninnhold (water) g/100 g 65.9

Nutritional Values, Water Content and Vitamins

Analysis by ALS (Germany) reveal that extracts may contain 60-70% water,100-200 kcal/100 grams, whereof 20-30 grams are proteins, 1-5 grams areash, and 0-2% are carbohydrates with sodium content from 0-1 gram/100grams; and a number of vitamins, including vitamin A and vitamin E

TABLE 3 Aske (ash) g/100 g 2 Protein g/100 g 26.4 Karbohydrater(carbohydrates) g/100 g 0.4 Energi (kiloJules) kJ/100 g 649 Energi(calories) kcal/100 g 154 Kostfiber (fiber) g/100 g 0.2 Na (sodium)g/100 g 0.22 Fett (fat) g/100 g 5.2 Vitamin A μg/100 g 10.7 Vitamin Emg/100 g 9.9

Summary of Physical Properties.

Preparation of extract from homogenates of salmon and trout eggs givedifferential separation of RNA, DNA and protein, but equal separation oflipids. Fertilized and unfertilized salmon egg extracts display the sameprofiles of protein, RNA and DNA.

1) The protein concentration (180-300 mg/ml) of the extract is roughlycomparable to that of the homogenate (no or little protein removed byproduction method regardless of g-force).

2) The RNA content (2-5 mg/ml) seems roughly equal for salmon and trouthomogenates, slightly lower in trout. RNA seems to be increased in theextracts made from trout egg homogenates, which may be due to the lesserviscosity and better solubility of RNA in the extract fraction fromthese eggs. RNA content in final salmon egg extracts is slightly lowerthan that of trout egg extracts.

3) The DNA content (40-500 ug/ml) of the extracts is highly variable,which is probably caused by differential lysing of nuclei in theegg-crushing homogenization process. Salmon egg extracts appear to havelower DNA content than trout egg extracts. In both extracts, and DNAcontent is lower in extracts than in homogenates, indicating that someDNA is spun down at g-forces over 9.379 g.

4) Total lipid content (3.7-4.5%) is roughly equal for salmon and troutextracts. It seems equal amounts of the lipids are separated from theextract fraction at most g-forces over 1700 g. At lower centrifugalforces which may be use to produce the extract, the total lipid contentmay be higher, 4-7%.

Example 8 Production of Extracts

It has been documented that lipid content of the extract surprisingly isunchanged at centrifugation speeds varying from 1,700 g to 15,000 g (seeabove), while other parameters such as RNA, DNA and protein content isaltered with the increase of g-force during centrifugation. An extractmay also be produced by lower centrifugal forces, down to 400 g,whereupon the content of especially lipids may be higher.

An extra step of washing the eggs for 10 minutes with buffodine (1:100in 0.9% NaCl) before preparation of homogenate is beneficial. Thiswashing step appears to reduce the bacterial content significantly. Forsafety reasons, all LEX batches packaged in final containers are mildlypasteurized (incubated) by heating to 56° C. for 20 minutes. Thispasteurization sterilizes the extract completely, with 0 bacteria foundin extracts plated on bacteria dishes incubated for 3 days at roomtemperature, 4 degrees centigrade or 30 degrees centigrade. 1 colony/100ul LEX plated on agar dish incubated at room temperature is the maximumobserved. This is 100× below safety limits for drinking water (100bacteria/ml). A single colony seldom observed probably comes from theair during the plating of LEX, and is comparable to bacterial growth ofnegative control (plate only).

The stability of LEX and collagen secretion effect is retained after LEXis heated to 56 C for 20 minutes. When applied to human fibroblasts invitro at 0.5% concentration in cell media for 8 days (media changeddaily), the effect on collagen secretion (as measured as efflux ofcollagen from cells to cell medium and compared to untreated controlcells), was comparable to cells treated with unheated extract which hadbeen kept at −80 C after preparation. A 200-400% increase compared tocontrols was observed for both heated and unheated LEX. Previously wehave seen a decreased effect on collagen secretion with extractsincubated at 72 C, indicating that active substances in the extractswhich may be denatured between 56 and 72 C are responsible for parts ofthe secretion effects. In this temperature range, proteins are known todenature. It may be deduced that a structured protein is one of theactive substances.

Example 9 Pilot Scale Production

This example describes the preparation of LEX extracts from fresh salmonor trout roe/eggs sent on ice overnight from a hatchery. Eggs that havebeen in transit on ice >48 hours are discarded. If the eggs can't beprepared on the day of arrival, the eggs may be stored at −20° C. for upto 12 months. The extracts are during lab scale production preparedusing an Avanti J-26 XP ultracentrifuge with a JLA 8.1000 rotor and6×1000 ml tubes (polypropylene, #363678 with liner). For pilot-plantscale production, the homogenate and extract are prepared using a 20litre Hydropress and a Separator, (OTC 3-03-107 Mineral oil centrifugerespectively.

The day before preparation of extracts, the separator components, glassand stainless steel equipment are autoclaved. Desired volume of PBS/0.9%NaCl and Buffodine (in 0.9% NaCl) are prepared according to the amountof used roe/eggs. The buffers are placed in a cold room.

Pilot-plant production: On the day of extraction, the hydropress (20, 40or 90 litre, Vigo, England) and separator OTC 3-03-107 (Gea Westfalia)are assembled according to the manufacturer's instruction. Silicon tubesare sterilized in 70% Ethanol. A coarse polypropylene press sack iswashed once in UV-sterilized water for 1 hour.

The materials and the production procedure are handled at roomtemperature. Thawed eggs are washed in buffodine for 10 minutes (1-2:100Buffodine in 0.9% NaCl) and drained. The eggs are rinsed 4× in distilledwater containing 0.9% NaCl. Eggs are homogenized (pressed) in theHydropress using a coarse polypropylene press sack. Homogenate iscollected in a sterile beaker after being sieved to remove egg shell anddebris. Egg shells and debris are discarded from sieve and Hydropress(all extract trash in biowaste). The homogenate is transferred from thebeaker to the separator through silicon tubes (2.4 mm wall thicknesswith an inner diameter of 8 mm 5/16″) using a peristaltic pump (WatsonMarlow 323 S with Pumphead 314D, VWR International). The homogenate isseparated in the separator with an internal pressure of 0.5-2.5 bar (thepressure inside the separator removes the fat fraction of the homogenateand reduces the total fat content in the homogenate from ca 7.2 to ca4.0-5.5%). The middle (cytoplasmic) fraction is collected in a beaker,and is thereafter transferred to freeze-resistant containers (eppendorftubes (1 ml aliquots), 8-strip PCR tubes (200 μl aliquots) and 500 mlcanisters. If desired, extracts can be pasteurized by heating at 56degree centrigrade for 20 minutes prior to freezing Extract aliquots arefrozen immediately at −80° C. and can be stored for up to one year. Theextracts have a pH of from 6.5-7.0; have a bacterial load of less <100colonies per ml (e.g. <10 colonies per plate) as tested onantibiotic-free agar plates; an osmolarity of from 300-500 mOsm; and aprotein content of from 100-300 mg/ml.

Extract properties from the lab scale- and pilot plant production areprovided in Table 4.

TABLE 4 Extract Properties Sample Lab scale production Pilet plantproduction Protein Nanodrop 192-203 Nanodrop 194-199 (mg/ml) Fluoremeter163-194 Flouremeter151-178 DNA (ug/ml) 14.7 17.9 Fat Homogenate (NA)Homogenate 7.2 (%) Lex 36 4.9 LEX 37 1 bar 4.9, LEX 37 2 bar 4.8 LEX 37total 5.0 mOsm 345-364 358-361 pH 6-7 6-7 Collagen effect 3-6 × foldinduction 4-5.5 × fold induction In test creme No smell No smell

Properties of extract produced in lab scale or pilot plant scale (in redletters). NA indicate—Not Analysed.

Example 10 Extract (LEX) Reverses Age Induced Loss of Collagen byStimulating Collagen Secretion

With age, collagen secretion from skin fibroblast decreases. Studieshave shown that the natural secretion of collagen decreases by 38% fromage 25 to age 80 ((Varani J. et al. Decreased collagen production inchronologically aged skin: roles of age-dependent alteration infibroblast function and defective mechanical stimulation. Am J Pathol.2006 June; 168(6):1861-8.).

Treating human skin fibroblasts (hSF) for 8 days with 0.5% of theextract in normal cell media increases the collagen secretion from thecells by 500%. See FIG. 6.

Example 11 Extract Treatment Increases the Proliferation of Fibroblasts

Treatment of human skin fibroblasts (hSF) with 0.5% extract added tonormal cell media increases the proliferation of the cells in vitro by40% in 7 days. See FIG. 7.

Example 12 The Extract Decreases the Appearance of Fine Lines In Vivo

Twenty-two test persons (of Asiatic origin) were divided into 3 groups,the study lasted 56 days, measurements of skin roughness were taken onday 0, 7, 14, 28 and 56. Asian skin usually responds less well towrinkle reducing treatments than Caucasian or black skin. Thesignificant effect seen with 5% extract in this study may mean a lower %extract may be necessary to have a significant effect in other skintypes.

A basic water based serum with 12% glycerol was used a base control(group 3), into which it was added 5% extract (group 1) or 1% extract(group 2).). Test persons applied the appropriate cream daily.

Surface roughness (SA) was measured with from PRIMOS_(—)3D system(Canfield, USA).

5% extract concentration (group 1) decreases the appearance of finelines by 20% compared to control. The effect is statisticallysignificant. The results for 5% extract shows a significant decrease insurface roughness is significant to control. The dose-response curveshow larger improvements over time. This study shows a remarkable,significant effect on the decrease of fine line in only 7 days overcontrol. Significant decrease in fine lines over control (group 3) arefound at day 7, 14 and 28. Extract at 1% (group 2) also showed asignificant effect over control on day 7. See FIG. 8.

This effect on the reduction of fine lines may be due to an increasedproduction in collagen in the skin and possibly an increase in thenumber of fibroblasts. Further in vivo studies involving biopsies wouldneed to be conducted to document this. The dose-response curve is inagreement with our in vitro results on fibroblast cells in culture,where collagen secretion increases with time, and is concentrationdependent.

However, as with retinoin, not all the volunteers have an effect, evenafter 56 days. Analyzing the raw data, it appears that for 5% extract,40% has an extremely good effect, 40% has a moderate effect and 20% hasno effect. This can be dependent on age, or the lack of vitamin C oriron, on which the formation of collagen is dependent or geneticfactors, or that the test person did not apply the serum as instructed.

According to others studies, only about 45% of women using retinoid-likecreams have an improvement after 1 year—the results seen with theextract are thus truly unique. To our knowledge there are no otherproduct tested this way that has such a large and significantimprovement in only 28 days as seen here with 5% extract.

Example 13 Extract Gives a Significant Reduction of the Melanin Index inSkin

Twenty-two test persons (of Asiatic origin) were divided into 3 groups,the study lasted 56 days, measurements of skin roughness were taken onday 0, 7, 14, 28 and 56. A basic water based serum with 12% glycerol wasused a base control (group 3), into which it was added 5% extract(group 1) or 1% extract (group 2). Test persons applied the appropriatecream daily.

Melanin index (MI) was measured with Mexameter (MX18, Courage+Khazaka,Germany). Measurements were taken at 5 areas of the face: forehead,outer edge of eye, cheekbone, outer edge of mouth and the chin.

The measuring principle for melanin and erythema readings is based on asource of light with three specific wavelengths whose radiation isabsorbed by the skin and diffusely reflected. A photo detector analysesthe diffuse reflection from the skin. It gives information onmicrocirculation in the skin and amount of pigmentation.

Increased melanin index is a measure of age spots, uneven skin tone andmay be caused by sun exposure and/or aging. A smooth and even skin toneis an important element in the perception of skin and a measure in skinyouthfulness.

Statistical analysis show that LEX 5% is significantly better atreducing pigmentation than group 3 (control) at all time points, Group 2(1% extract) is significant to control at day 7, 14 and 56.

There is an additional significant increase in the effect betweentimepoints within Group 1, starting at day 14 significant to day 0 andimproving throughout. Day 56 significant to day 0 and day 7. See FIG. 9.

Example 14 Improvement of Erythema Index—Reduced Redness of the Skin

Twenty-two test persons (of Asiatic origin) were divided into 3 groups,the study lasted 56 days, measurements of skin roughness were taken onday 0, 7, 14, 28 and 56. A basic water based serum with 12% glycerol wasused a base control (group 3), into which it was added 5% extract(group 1) or 1% extract (group 2).). Test persons applied theappropriate cream daily.

Erythema index (EI) was measured with Mexameter as with MI, giving ameasure of the redness of skin. Measurements were taken at 5 areas ofthe face: forehead, outer edge of eye, cheekbone, outer edge of mouthand the chin. Red, uneven skintone may be caused by skin irritation ormicrocirculation becoming visible (varicose veins) in thinning, agingskin.

Group 1 (serum with 5% extract) is the only of the only group to have asignificant effect. Significant improvement (less redness of skin) isfound at day 56 compared to day 0, 7 and 14. See FIG. 10.

Example 15 Improvement in Skin Lucidity, Lightness and Skin Coloration

Twenty-two test persons (of Asiatic origin) were divided into 3 groups,the study lasted 56 days, measurements of skin roughness were taken onday 0, 7, 14, 28 and 56. A basic water based serum with 12% glycerol wasused a base control (group 3), into which it was added 5% extract(group 1) or 1% extract (group 2).). Test persons applied theappropriate cream daily.

L*a*b value measures with a Spectrophotometer (CM2600d, Minolta, Japan),giving a measurement of how light and lucent the skin is. Measurementswere taken at 5 areas of the face: forehead, outer edge of eye,cheekbone, outer edge of mouth and the chin.

Group 1 (5% extract) gives a significant improvement with increased skinlucidity/lightness. 5% extract gives a significant effect on skinlucidity over all the other groups at day 14. 1% extract (not shown) isalso significantly better than control on day 7. See FIG. 11.

Example 16 Improvement in Skin Hydration and Skin Water Content andImprovement in Skin Sebum Levels

Twenty-two test persons (of Asiatic origin) were divided into 3 groups,the study lasted 56 days, measurements of skin roughness were taken onday 0, 7, 14, 28 and 56. A basic water based serum with 12% glycerol wasused a base control (group 3), into which it was added 5% extract(group 1) or 1% extract (group 2).). Test persons applied theappropriate cream daily.

Measurement of water content in skin by Corneometer (CM825,Courage+Khazaka, Germany). This is an analysis of the moisture retentioncapacity of the skin, based on the dielectric constant of the water andmeasured in the superficial layers of the stratum corneum as deep as10-20 μm to ensure that the measurement is not influenced by capillaryblood vessels. Measurements were taken at the cheek area.

Serum with 5% extract (group 1) improves water content in skinsignificantly better than the other groups at all time points. See FIG.12.

Measurements of skin sebum content with Sebumeter (SM810,Courage+Khazaka, Germany) show a decrease in sebum content in the skin,which is beneficial in conditions where skin has excess amounts ofsebum. Such a decrease may also indicate that skin has higher watercontent compared to sebum, which indicates better skin hydration.Measurements were taken at the chin area. See FIG. 13.

Example 17 Evaluating the Melanin Production in Human EpidermalMelanocytes (HEM) after Stimulation with Different Concentrations of LEX

Materials:

HEM cells, 25 cm² culture flasks, LEX 42 (Salmon roe extract), Culturemedium: Medium 254 with 1% HMGS and 1% PenStrep, Trypsin-EDTA solution,1M NaoH with 10% DMSO for cell lysis, Nanodrop for absorbancemeasurement.

Procedure:

Day zero remove the culture medium of one 175 cm² flask with HEM cell.Add 4 ml of trypsin-EDTA solution until cells are dispersed (1-2minutes). Add 8 ml culture medium and aspirate cells by gently pipettingup and down. Seed cells in 25 cm² flasks at a density of 7×10̂5 perflask. On day one, replace culture media with fresh media supplementedwith 0.5%, 1% LEX or normal culture media for control cells. Continuethe cultures for seven days, with a media (LEX/Control) change every 24hours. On day nine, wash the cells with PBS and add 0.5 ml trypsin-EDTAto each 25 cm² flask, count the cells before lysing them in 1M NaOH with10% DMSO at 80° C. for 2 hours. Spin down the cell debris (13,400 rpm,10 min) and measure the absorbance of supernatant at 470 nm using theNanoDrop 2000c (Thermo).

Results:

The results showed a decreased proliferation rate in LEX stimulatedmelanocytes compared to control cells. After eight days of LEXstimulation there were 1.09×10̂6 cells per flask treated with 0.5% LEX(33% reduction compared to control), there were 1.12×10̂6 cells per flasktreated with 1% LEX (32% reduction compared to control), see table 5.The decrease in cell number seen in LEX stimulated cultures demonstratethat LEX reduces the proliferation rate of melanocytes.

The melanin levels were measured for all three conditions after eightdays. The results demonstrate that the melanin production in total perflask is lower in the cultures stimulated with LEX compared to controlcells, but the numbers of cells were, as mentioned above, also lower inthe stimulated cultures than in the control cultures. The melanincontent per cell in the cultures treated with 0.5% LEX was 0.7 fold thatof control cells, a 30% decrease. For the culture treated with 1% LEXthe melanin per cell was 0.82 fold that of the control, an 18% decrease,(following Table and FIG. 14).

These results demonstrate that LEX slows down the proliferation rate ofmelanocytes, which further leads to a decreased level of melaninproduced per cell. In skin this process leads to a lighter skintone/pigmentation after LEX treatment.

TABLE 5 Overview of cell number, melanin content and melanin per cellafter eight days of LEX/control stimulation of melanocytes cells.average final cell SEM # of t-test # of abs →μg Average ng Fold SEMT-test Sample # cells cells melanin melanin/cell change (ng/cell)(ng/cell) 0.5% LEX 1096030 18797 2.09642E−05 12.09 0.0105 0.70 0.0020.94098677 0.5% LEX 7.545 0.5% LEX 14.82 1% LEX 1121900 485720.000497534 12.09 0.0124 0.82 0.0006 0.018370876 1% LEX 15.73 1% LEX13.91 Control 1645410 14641 25.73 0.0151 1.00 0.0004 Control 24.82Control 23.91

Example 18 Comparing the Effect of Retinoic Acid with LEX Stimulation onHuman Skin Fibroblasts In Vitro

To compare the in vitro effects of retinoic acid versus LEX on collagenproduction in human skin fibroblasts.

Materials:

hsF cells, 25 cm² culture flasks, LEX 42 (salmon roe extract), retinoicacid (RA) dissolved in ethanol, lx PBS at room temperature, Biocolor kitto assay collagen content in medium, culture medium: DMEM with 10% FBSand 1% PenStrep, starvation medium: DMEM with 1% FBS and 1% PenStrep.

Procedure: LEX 42 and different concentrations of retinoic acid (0.01Mand 0.1M) are applied to fibroblasts (HsF cells) in vitro and collagenproduction and secretion is measured. Briefly, on day 0, fibroblasts areseeded in 25 cm² cell culture flasks at a density of 2.8×10⁵ cells perflask. On day 1, the culture media is replaced with fresh culture mediasupplemented with 0.5% LEX, 0.01M RA, or 0.1M RA. Control cells aregrown with cell culture medium without LEX or RA. The cultures arecontinued for seven days, with a media (LEX/RA/control) change at day 4.On day 8, the cells are washed 3× with PBS before adding cell culturemedium with low serum (1% FBS). On day 9, the culture medium (1.4 ml) isharvested and collagen content assessed using a collagen kit (Biocolor,on eht world wide web at biocolor.co.uk/index.php/assay-kits/sircol-1/).The absorbance is measured with Nanodrop 2000c (Thermo).

Results:

After stimulating hsF cells with LEX 42 for eight days a 1.66 foldincrease of collagen/cell was found in the stimulated cell mediumcompared to control. The collagen produced per cell in the RA stimulatedcultures showed a decrease compared to LEX stimulated cells and control,the RA cultures showed a 0.84 and 0.89 fold change compared to controlcultures, (see following Table, and FIGS. 15 and 16). This correspondsto a 16.5% (p<0.31) decrease of collagen produced per cell for the cellsstimulated with 0.01M RA, a 11.5% (p<0.31) decrease of collagen per cellfor the cells stimulated with 0.1M RA and a 65.8 (p<0.011) increase forthe cells treated with LEX, compared to control.

The result showed a higher proliferation rate in the cultures stimulatedwith LEX, compared to both control and RA stimulated cells (table 6).LEX stimulated cells showed a 23% increase (p<0.024) in cell numberafter 8 eight days compared to the control cultures. For the 0.1M RAtreated cells a 10% (p<0.5) increase in cell number, compared tocontrol, was observed, and for the 0.01M RA treated cell only a 3.9%(p<0.33) increase in cell number was observed.

These results shows that RA does not induce hsF cells to produce morecollagen, which could fill and reduce fine lines/wrinkles, in vitro inthe same manner as LEX. No increase in collagen production of hsF cellswere seen as a result of stimulation by RA in this experiment.

TABLE 6 Overview of initial and final cell numbers, collagen absorbancevalues, collagen per cell and percentage change between the treatmentregimens LEX, 0.1M RA, 0.01M RA and control cultures. Fold Cell %Coll/cell % Sample initial # of cells final # of cells Collagen AbsCollagen/cell induction change change 3x 0 μM RA 280429 871667 0.0505.73614E−08 1.00 0 0 (control) 3x 0.01 μM RA 280429 959833 0.046 4.7925E−08 0.84 10.1 −16.5 3x 0.1 μM RA 280429 906000 0.046 5.07726E−080.89 3.9 −11.5 3x LEX 42 280429 1072333 0.102 9.51197E−08 1.66 23.0 65.8

Example 19 Modification of Collagen Assay to Avoid Overestimation ofCollagen Secretion from Fibroblasts Caused by Serum Proteins and LEXRemnants

Materials:

hsF cells, 25 cm² culture flasks, LEX 51 (salmon roe extract), lx PBS atroom temperature, Biocolor kit to assay collagen content in medium,culture medium: DMEM with 10% FBS and 1% PenStrep, starvation medium:DMEM with 1% FBS and 1% PenStrep.

Procedure:

LEX 51 was applied to fibroblasts (HsF cells) in vitro, and collagenproduction and secretion was assayed. The procedure was as describedabove, except with the modification of the assay on day 10, were 2 ml ofthe culture medium is harvested and collagen content assessed using acollagen kit (Biocolor, biocolor.co.uk/index.php/assay-kits/sircol-1/),with the adaptation of isolating collagen from 2 ml of culture mediumcompared to 1 ml as described in the kit manual. The absorbance ismeasured at 550 nm (the base line was set to 750 nm) with the Nanodrop2000 (Thermo)

Results:

The results show a 3.342 fold increase of collagen present in the mediumof LEX stimulated cells compared to the control cells. The results showthat the fold induction is significant with p<0.025 (t-test, tail 2),see table 1. The figure shows a 3.42 fold induction of collagen in themedium of LEX stimulated cells (0.167) compared to control (0.049), thedata in FIG. 1 are presented as an average (n=3)±SEM (standard error ofthe mean) t-test: p<0.025.

TABLE 7 Collagen absorbance of hsF cell medium stimulated with LEX 51and non-stimulated control cells. Triplets of the experiment were runfor both stimulated and control cells Sample ID Date and Time 1 (nm) 1(Abs) abs × 2 ml Average Fold Induction SEM t-test Control 08 Mar. 201114:44:33 550 0.012 0.024 0.049 1 0.006 Control 08 Mar. 2011 14:45:14 5500.029 0.058 Control 08 Mar. 2011 14:45:31 550 0.032 0.064 51 08 Mar.2011 14:47:29 550 0.076 0.152 0.167 3.42 0.015 0.024 51 08 Mar. 201114:47:49 550 0.061 0.122 51 08 Mar. 2011 14:48:08 550 0.113 0.226

Example 20 Evaluation of Melanin and Collagen Production in In VitroSkin Model Including Human Skin Fibroblasts, hsF (ATCC) and HumanEpidermal Melanocytes, HEM (Invitrogen)

Materials:

hsF cells, culture flasks (25 cm²), HEM cells, LEX 51 (salmon roeextract), Culture medium for HEM cells (Medium 254 with Human melanocyteGrowth Supplement, HMGS (Invitrogen), medium for hsF cells (DMEM with10% FBS and 1% PenStrep), culture medium for co-culture of both hsF andHEM cells (Medium 254 with 10% FBS and 1% PenStrep), 1×PBS at roomtemperature, TE to loosen cells from culture flasks, biocolor kit toassay collagen content in medium.

Procedure:

hsF cells are prepared as described above. HEM cells are prepared asdescribed above, and seedHEM cells (200 000/flask) on top of hsF cells.On day two, the culture media is replaced with fresh media supplementedwith 0.5% LEX. Control cells were grown in medium without LEX. Theculture is continued for seven days, with the media (LEX/control)changed every 24 hours. On day eight, the cells are washed 3× with PBSand medium with low serum is added. On day nine the collagen content ofthe cell medium is measured by using the sircol collagen assay(Biocolor) and measuring the absorbance against a standard curve ofcollagen. The level of melanin produced by melanocytes is measured bylysing the cells in 1M NaOH at 80° C. for 2 hours before spinning downthe cells at 12,000 rpm, for 10 min(minispin) before measuring theabsorbance of the supernatant at 470 nm (Nanodrop 2000c) against astandard curve of melanin.

Results:

The collagen production in co-culture was similar to what was seen inhsF cells grown in monoculture, see Table below. The comparison was notrealistic as the monoculture assay was performed without the isolationand concentration step, making the results from the monoculture assayoverestimated, due to serum and LEX proteins present in the mediumduring the staining procedure.

LEX stimulation of co-cultured hsF and HEM cells resulted in a higherrate of collagen production/secretion into the medium than for controlcells (FIG. 18). A 1.57 (p<0.02) fold increase (Table 8) of collagen inthe medium of co-cultured hsF cells was seen. This fold induction wasrealistic as the collagen is isolated from the background of serum andLEX proteins which is shown to bind the assay dye and contributes to anoverestimated collagen level. The collagen levels measured formonocultures of hsF cells stimulated with LEX 49 showed a 1.69(p>0.0002) fold induction of collagen compared to control, but in thisexperiment collagen was not isolated from neither LEX nor serumproteins, making the obtained absorbance values an overestimation ofcollagen present in both stimulated and control medium. These resultsindicate that co-culturing hsF and HEM cells did not interrupt norchange the hsF cells' ability to increase production of collagen uponLEX stimulation.

The level of melanin in the HEM cells was also measured after 8 days ofstimulation with LEX 49. When comparing the co-culture melanin levels tomelanin measured in melanocytes grown in monoculture, both stimulatedand control cells, the results show a higher level of melanin per cellin co-cultured cells than in mono-cultured cells. For the co-culturedHEM cells, 0.028 ng/cell after LEX stimulation and 0.026 ng/cell forcontrol (Table 9, FIG. 19) compared to 0.015 ng/cell in mono-culturedLEX treated cells and 0.011 for non-treated mono-cultured cells. Thismay demonstrate that melanin production is stimulated/up regulated byfactors secreted by fibroblast cells when grown together with HEM cells.

With the co-culture experiment, as seen with mono cultures ofmelanocytes, LEX affected the proliferation/division of the melanocytes.When stimulating melanocytes with LEX, the cells did not divide asactively as in the control cultures, there were fewer cells after eightdays of LEX treatment, 5.87×10̂5 cells/flask, compared to 6.99×10̂5cells/flask in the control (table 3). This was also observed formono-cultured HEM cells, 1.1×10̂6 cells per flask compared to 1.64×10̂6cells per flask after eight days. All in all an in vitro model forco-culturing fibroblast and melanocytes has been established, a methodthat can be used for mimicking the intact skin.

TABLE 8 Collagen production of hsF in co-culture with HEM cells and innormal monoculture (below double line). Sample ID Date and Time 1 (nm) 1(Abs) Average absorbance fold induction SEM T-test (p) Controlco-culture 27 Feb. 2011 15:39:43 540 0.079 0.083 1 0.004 Controlco-culture 27 Feb. 2011 15:40:01 540 0.091 Control co-culture 27 Feb.2011 15:40:18 540 0.079 LEX 49 co-culture 27 Feb. 2011 15:40:53 5400.109 0.131 1.57 0.012 0.0203 LEX 49 co-culture 27 Feb. 2011 15:41:17540 0.132 LEX 49 co-culture 27 Feb. 2011 15:41:35 540 0.151 LEX 49 25Nov. 2010 13:41:52 540 0.508 0.480 1.69 0.015 0.0002 LEX 49 25 Nov. 201013:42:14 540 0.476 LEX 49 25 Nov. 2010 13:42:35 540 0.457 Control 25Nov. 2010 13:36:41 540 0.278 0.284 1.00 0.004 Control 25 Nov. 201013:37:08 540 0.282 Control 25 Nov. 2010 13:37:33 540 0.291

TABLE 9 The level of melanin of the co-cultured melanocytes after 8days. Sample ID Date and Time 1 (nm) 1 (Abs) abs →μg melaninMelanin/cell, ng/celle Control co-culture 26.02.2011 10:58:16 470 0.02823.00 0.034 Control co-culture 26.02.2011 10:58:54 470 0.024 19.36 0.027Control co-culture 26.02.2011 10:59:21 470 0.021 16.64 0.023 LEXco-culture 26.02.2011 10:59:56 470 0.017 13.00 0.023 LEX co-culture26.02.2011 11:00:18 470 0.018 13.91 0.024 LEX co-culture 26.02.201111:00:37 470 0.024 19.36 0.031 Control monoculture 07.12.2010 470 0.03125.73 0.011 Control monoculture 07.12.2010 470 0.030 24.82 0.007 Controlmonoculture 07.12.2010 470 0.029 23.91 0.014 LEX monoculture 07.12.2010470 0.016 12.09 0.016 LEX monoculture 07.12.2010 470 0.011 7.545 0.015LEX monoculture 07.12.2010 470 0.019 14.82 0.015

Example 21 Collagen Production in In Vitro Skin Model Composed ofSeveral Known Skin Cell Types Including Human Skin Fibroblasts, hsF,(ATCC) and Human Epidermal Keratinocytes, HEK and Evaluating theCollagen Production

Materials:

hsF cells, culture flasks (25 cm²), HEK cells, LEX 51 (salmon roeextract), Culture medium for HEK cells (Eplife with Human KeratinocyteGrowth Supplement, HMGS (Invitrogen), medium for hsF cells (DMEM with10% FBS and 1% PenStrep), culture medium for co-culture of both hsF andHEK cells (Eplifie with 5% FBS, HKGS and 1% PenStrep), 1×PBS at roomtemperature, Trypsin-EDTA to loosen cells from culture flasks, Trypsinneutralizer (Invitrogen) and biocolor sircol kit to assay collagencontent in medium.

Procedure:

hsF cells were prepared as above and allowed to attach for 24 hours. HEKcells were prepared as described and seeded on top of the hsF cells (200000 cells/flask). On day two, replace the culture media with freshco-culture media supplemented with 0.25% LEX. Control cells are grownwith cell co-culture medium without LEX). Continue the culture for sevendays, with a media (LEX/control) change every 24 hours. On day eight,wash the cells 3 times with PBS and add medium with low serum. On daynine, assay the collagen content of the cell medium using Sircolcollagen assay (Biocolor).

Results:

Co-culture of hsF and HEK cells

In earlier experiments LEX has been tested on monoculture of HEK cells,resulting in all the cells dying within 2-4 days. The lower theconcentration of LEX added to the culture medium, the longer the cellssurvived. When co-culturing HEK cells with fibroblasts, however, theykept a normal morphology as well as looking healthy while beingstimulated with 0.25% LEX for eight days.

The results show that fibroblasts and keratinocytes can be co-culturedin the medium mentioned above, as well LEX is not toxic forkeratinocytes when co-cultured with fibroblast cells.

Fibroblasts in co-culture with HEK cells produced collagen at acomparable level to when grown in monoculture (table 4), demonstratingan established model for co-culturing keratinocytes and fibroblasts.

TABLE 10 Collagen concentration of the medium of hsF and HEK cellsco-cultured with LEX stimulation for eight days before allowing cells tosecrete collagen into medium (low serum, 1%) for 24 hours. Sample IDDate and Time Collagen concentration (ug) co-culture LEX 24.03.201113:55:37 6.89 co-culture LEX 24.03.2011 13:55:58 8.85 co-culture LEX24.03.2011 13:56:17 9.09

Example 22 Stem Cell Proliferation Assay: An In Vitro Model Studying theEffect of LEX on Stem Cell Proliferation Materials: Cells

-   -   ADSC adipose derived stem cells at passage up to 25

Kit

-   -   Cell proliferation kit XTT, Applichem

Medium and Solutions

-   -   1×PBS, phosphate buffered saline    -   DMEM, Dulbecco's modified eagle medium, Sigma    -   FBS, Fetal bovine serum, Sigma    -   P/S, Penicillin Streptomycin, Sigma

Material

-   -   96 well plates, Corning Costar    -   Nanodrop 2000c, Thermo scientific

Methods:

LEX 51 extract prepared as described earlier is applied toadipose-derived stem cells (ADSC) in vitro and proliferation rate ismeasured and compared to un-stimulated control cells. 5000 ADSC areseeded per well, in 96 well plate. 2 days after seeding the culturemedia is replaced with (100 μl per well) fresh culture media added 0.1,0.25, 0.5 and 1.0% LEX. Control cells are cultured with (100 μl perwell) cell culture medium without LEX. The cell cultures are added freshmedium every 24 hours (with or without LEX). Proliferation rate ismeasured at day 3, 6 and 9, using the cell proliferation kit XTT fromApplichem. XTT reagent- and activation solution is prepared as describedin Applichem product sheet (on the world-wide web atapplichem.com/en/shop/product-detail/as/zellproliferations-testkit-xtt/).Three culture wells from control and stimulated cells is analyzed on day3, 6 and 9. Before measuring absorbance (high absorbance demonstratehigh proliferation and vice a versa) each well is washed several timesusing PBS, to remove excess LEX and FBS. This is because LEX retains thecolor from the proliferation assay giving overestimated measures. XTTreaction solution (50 μl) is added to each of the wells, the cultureplate is afterwards incubated for 8 hours. Shake the plate gently todistribute the dye in each well, 10 μl of the dyed medium is thenextracted for analyzing the absorbance. One blank sample is alsoprepared (100 μl fresh medium and 50 μl XTT reaction solution). Theabsorbance is then measured using Nanodrop 2000c from Thermo scientific.Wavelengths of 450-500 nm is used to measure, the background withwavelengths of 630-690 nm are subtracted.

Results:

To evaluate the optimal time point for reading the absorbance with theXTT proliferation assay on ADSC cells, an analysis was done. Byextracting samples every two hours after adding reagent solution andplotting these against absorbance, an optimal time point for reading wasfound; after 8 hours. Results from this analysis are shown in FIG. 20.

FIG. 20 show absorbance readings over 2-12 hours with ADSC stimulatedand control cells at day 3. Cells stimulated with 0.1%, 0.25%, 0.5% and1.0%, and control cells were analyzed. It was found during this testingthat it is necessary to add a washing step with PBS during the assay toremove LEX and FBS. FIG. 1 show lower proliferation rate with higher LEXconcentration stimulation, this was found not to be the case since LEXretained color in the absorbance assay. Values were observed tostabilize after 8 hours of reaction time. Henceforth absorbance readingwas done 8 hours after adding XTT reaction solution.

Following this analysis a complete proliferation assay was done asdescribed above but also adding the washing step. The proliferationassay were done both with 1% and 10% FBS to evaluate whether FBScontains signaling molecules that increase the proliferation rate inADSC. Result from the complete proliferation assay is given in FIG.21, * P<0.05 and ** P<0.05 for 0.5% and 1.0%

Results from proliferation assay using 10% FBS are given in FIG. 22.FIG. 22 show a similar shape as seen with 1% FBS with the highestabsorbance at day 6 of stimulation. The values of stimulated samples onday 6 is not significant compared to control. At day 9, the 0.1%, 0.25%and 1.0% LEX stimulated samples are significant compared toun-stimulated samples.

The proliferation assay results were not unambiguous concerningsignificant values in 1% FBS and 10% FBS. This would indicate that FBSmight affect the proliferation rate in ADSC. Still the general trend ofthe 3, 6 and 9 days samples was still roughly the same with the highestabsorption values after 6 days of stimulation, demonstrating that LEXincrease the proliferation rate in ADSC.

Example 23 Microarray Analysis

Micro array data of fibroblasts stimulated 8 days with three differentbatches of extract (LEX 40, 42, 43) the three different groups are notsignificant different from each other, confirming that the differentbatches of the extract produces the same up or down regulation of genes.The micro array data of fibroblasts show up/down regulation of more than550 genes compared to untreated cells. These genes include but are notlimited to several genes previously shown to be involved in the processwhich is related to wound healing/cell regeneration/cell proliferation.Examples of genes regulated by the extract are; extra cellular matrixproteins like collagen I, III and V; syntases like hyaluronan (HAS2);enzymes like matrix metalloproteinases (MMP3) important for ECMremodeling and members of the kinesin family, important for translatingATP into mechanical work; signaling proteins like interleukin 13receptor and suppressor of cytokine signaling (SOCS1); proteinsimportant for proliferation like discs, large (Drosophila)homolog-associated protein 5 (DLGAPS) and cyclin A2 (CCNA2)

TABLE 11 Examples of gene regulation in human skin fibroblasts (hSF)after extract treatment Symbol FC Reg Biological process PDK4 2.78 upCarbohydrate/glucose/pyruvate metabolic process MYBPH 2.65 up celladhesion, regulation of striated muscle contraction ANGPTL4 2.47 up Celldifferentiation/neg regulation of apoptosis/pos reg ofangiogenesis/signal transduction HMOX1 2.26 up wound healing involved ininflammatory response/anti apoptosis/+++++ PLIN2 1.99 up Cellular lipidmetabolic process/lipid storage CYP26B1 1.96 up Cell fatedetermination/neg reg of RA receptor signaling patway/retinoic acidcatabolic process FBXL5 1.87 up Protein ubiquitination FOS 1.83 upAging, inflammatory response, innate immune resp IL13RA2 1.83 upcytokine mediated sign pathway/signal transuction SOX18 1.79 up haircycle/folllicle development, CDC20 1.79 up Cell cycle controler/checkpoint THBD 1.75 up Blood coagulation DLGAP5 1.76 up Cell cycle,proliferation, cell-cells signaling RSPO3 1.69 up Response to stimulus,wnt receptor signaling patway PLK4 1.66 up protein phosphorylation CENPM1.64 up Cell cycle (mitotic) CEP55 1.66 up Cell division GTSE1 1.66 upG2 phase of mitotic cycle/DNA damage response induced by p53 resultingin cell cycle arrest MAD2L1 1.66 up Cell division check point proteinCHTF18 1.63 up Cell cycle/DNA replication CDC25C 1.67 up Cellcycle/division, cell proliferation, DNA replication, regulation of cellcycle PTTG1 1.63 up Cell cycle/division, negative reg of prolif, reg ofcell growth PENK 1.62 up Behavior, nueropeptide sig path, perception ofpain TACC3 1.61 up Cell proliferation, regulation of cell cycle TPX2 1.6up Activation of protein kinase acivity, apoptosis, cell cycle/division,KIF4A 1.61 up Axon guidance, blood coagulation KIF20A 1.6 up Microtubulebased movement, m phase of mitiotic cycle, protein transport TROAP 1.58up Cell adhesion ASPM 1.58 up Cell cycle/divsion, negative reg ofsymmetric division CDCA8 1.58 up Cell cycle/divsion, HJURP 1.57 up Cellcycle CDC25B 1.56 up Cell cycle/division, pos reg of protein kinaseactivity FAM83D 1.57 up Cell cycle/division CENPF 1.55 up Celldifferentiation, cycle/divsion, proliferation, reg of muscle tissuedevelopment, PKMYT1 1.57 up cell cycle, phosphorylation, kinase activityANLN 1.55 up Cell cycle, mitosis, SPC24 1.54 up Cell cycle/division,mitosis DEPDC1 1.55 up Intracellular signal transduction TYMS 1.54 upAging, cartilage development, organ regeneration, cell cycle/regulationof transcription, DDIT4L 1.53 up negative regulation of signaltransduction TK1 1.52 up DNA replication, liver development, response tonutrient levels, CENPE 1.53 up microtubule based movement, pos reg ofprot kinase activity MELK 1.51 up Protein phosphorylation NCAPG 1.51 upchromosome condensation CCNB1 1.49 up Cell cycle checkpoint/divisionPLCXD3 1.46 up lipid metabolism and intracellular signaling C1QTNF6 1.47up CCNF 1.45 up Cell cycle/division RASD2 1.43 up Signal transductionCDCA3 1.44 up CCNA2 1.43 up positive regulation of fibroblastproliferation, cell cycle/divsion KIF18A 1.42 up MMP3 1.41 up KIF11 1.43up KIF14 1.41 up UCP2 1.41 up Electron transport chain, response toglucose, fatty acids, hormones/insulin., protein transport SOCS1 1.41 upsignaling patway, fat cell differentiation, regulation of growth RFC31.41 up cell cycle check point, telomere maintenance NCAPD2 1.4 up cellcycle/division COL1A1 1.4 up ANGPTL4 1.42 up cell differentiation, lipidmetabolism, neg reg of apoptosis, pos reg of angiogenesis NFIL3 1.4 upimmune response CCNB2 1.39 up cell cycle/division KRT14 1.38 upepithelial cell differentiation, keratin fillament, epidermisdevelopment ITGA3 1.38 up cell adhesion, blood coagulation FAH 1.36 upamino acid metabolism ITGA5 1.36 up cell adhesion, blood coagulation,leukocyte migration, WOUND HEALING, pos reg VEGF patway PGF 1.34 up cellcell signaling, angiogenesis, cell division, pos reg proliferationCOL3A1 1.34 up CSPG4 1.34 up ECM molekyl, angiogenesis, differentiationHMMR 1.33 up migration and transformation of fibroblasts, bindshyaluronic acid

Example 24 Milling process

Materials and Methods:

The mill consists of two metal-milling rolls produced by Monster BrewingHardware. One of the rollers is idle while the other roller is driven bya drive-shaft for drill. These are connected to a holder with adjustmentscrews for adjusting gap size between the rolls ˜0.5 mm to 2 mm. Aspecially made “hopper” is placed on top of the mill, working as afunnel for containing the roe and directing them directly to the mill.The top length and width are 322 mm and 290 mm, respectively. The loweropening has the dimensions, 147 mm and 18 mm.

Frozen, unfertilized salmon (Salmo salar) eggs are thawed at roomtemperature in Type 1 quality water produced by Milli-Q system. Thewater used for thawing is added NaCl (1:100 in 0.9%). Thawed eggs isthen sieved and washed in Type 1 quality water produced with 0.9% NaCland 2% buffodine from Evans Vanodine International Plc. Buffodine is aniodine complex fish egg disinfectant. Buffodine is effective againstviruses, bacteria and fungi. Roe is washed in buffodine solution for ≧10minutes. When finished washing, the eggs are cleaned using Type 1quality water with NaCl (1:100 in 0.9%) and sieved again. Duringthawing, washing and cleaning steps the eggs is covered with water.About 3-4 kg sieved eggs are then added to the hopper. The drill thenruns at a constant, speed (100-150 Rpm) running the rolls to homogenizesalmon roe. The homogenate gather in a sieve, retaining the solids (roeshell) while liquid gathers in a food-grade container. The remainingsteps of the production procedure are as described earlier.

The mill has been implemented in the production procedure since batch47.

Results:

The production procedure is now more efficient and retains the sameeffect on collagen production and secretion from hSF (fibroblast), whichis used as an effect control for LEX. The results are seen in Table 2.

Quality Control:

Protein concentration, pH and bacterial content was measured usingNano-drop (ND-1000 Spectrophotometer, NanoDrop Technologies, Inc.),pH-indicator paper, Merck and Caso agar plates from VWR, respectively.The results from quality control on LEX 47 to 51 are shown in Table 1.

TABLE 12 Quality control on LEX batches 47-51, showing species,preparation method, pH, protein concentration, as well as bacterialcontent in colony- forming units (CFU). Bacterial content LEX# mg/ml(CFU/ Species Preparation correspond to pH protein 100 μl) Salmon Maltmill and LEX 47 6.4 179 1.3 Salmon separator at 0-0.5 LEX 48 6.4 201 2.4Salmon bar pressure LEX 49 6.4 182 0.0 Salmon LEX 50 6.4 255 0.1 SalmonLEX 51 6.7 369 0.0

The pH measurements on batch 47 to 51 show little variation and are allwithin the predetermined acceptable values. The protein concentration isvarying between batches going from 179 up to 369 mg/ml. Concerningbacterial content, none of the batches were outside of the predeterminedcriteria for sale. Showing that heat treatment at 65° C. removesbacteria from the extract.

TABLE 13 Quality control testing on LEX 47-51, showing species,preparation method and fold induction on collagen production andsecretion. LEX# correspond Fold induction, collagen Species Preparationto production and secretion Salmon Malt mill and LEX 47 3.3 X Salmonseparator at LEX 48 7.8 X Salmon 0-0.5 bar pressure LEX 49 6.3 X SalmonLEX 50 3.0 X Salmon LEX 51 3.9 X

Collagen assay from Biocolor was used to analyze the effect of LEX onhSF (fibroblast). This assay measures the produced and secreted collagenfrom hSF (fibroblasts). The measurements from un-stimulated control wascompared to LEX stimulated hSF (fibroblasts) and noted as foldinduction. All collagen assay measurements from LEX 47-51 are within thepredetermined quality specification.

Example 25 Content Analysis on LEX Batches Fat Content- and ProfileAnalysis

Under production of LEX, a small fraction of fats remains in theextract. Tests have shown that remaining fats do not compromise theproduct. To confirm a stabile fat content and fat profile between LEXbatches, samples have been analyzed by ALS laboratory Group Norway AS,specializing in analytical chemistry and testing services.

A fat analysis was run to find eventual differences between LEX batches46 to 48 plus 42, shown in Table 1.

TABLE 14 Fat content- and profile analysis of LEX 46, 47, 48 and 42 doneby ALS laboratory Group Norway AS LEX 42 LEX 46 (Hydro (Hydro LEX 47 LEX48 ELEMENT SAMPLE press) press) (Mill) (Mill) Fett g/100 g 5.2 5.3 5.15.4 Fatty acids, saturated g/100 g 1.6 1.2 1.1 1.1 Fatty acids,monounsaturated g/100 g 2.8 2.6 2.7 2.8 Fatty acids, polyunsaturatedg/100 g 0.8 1.5 1.3 1.6 Transfettsyrer g/100 g 0 0 0 Sum omega-3fettsyrer g/100 g 0.9 0.8 1 Sum omega-6 fettsyrer g/100 g 0.5 0.5 0.6C4:0 Butyric acid g/100 g <0.10 0 0.042 0 C6:0 Caproic acid g/100 g<0.10 0.044 0.061 0 C8:0 Caprylic acid g/100 g 0.25 0.05 0.15 0.049C10:0 Capric acid g/100 g 0.62 0.031 0.067 0.028 C11:0 Undecanoic acidg/100 g <0.10 0 0.026 0 C12:0 Lauric acid g/100 g <0.10 0.15 0.11 0.16C13:0 Tridecanoic acid g/100 g <0.10 0 0.016 0 C14:0 Myristic acid g/100g 4.2 3.1 2.7 3 C14:1 Myristoleic acid g/100 g 0.15 0.046 0.047 0.046C15:0 Pentadecanoic acid g/100 g 0.34 0.33 0.3 0.31 C15:1cis10-Pentadecanoic acid g/100 g 0.12 0 0 0 C16:0 Palmitic acid g/100 g14.3 12.1 10.9 10.4 C16:1 Palmitoleic acid g/100 g 9.6 7.6 5.7 6.7 C17:0Heptadecanoic acid g/100 g 0.21 0.29 0.28 0.27 C17:1 Heptadecenoic acidg/100 g <0.10 0 0 0 C18:0 Stearic acid g/100 g 5.4 5.5 4.5 3.9 C18:1Oleic acid g/100 g 33.8 33.9 40 37.2 C18:2 Linoleic acid (omega6) g/100g 5 6.8 9.3 8.7 C18:3 Linolenic acid (omega6) g/100 g 0.47 0.071 0.120.1 C18:3 a-Linolenic acid (omega3) g/100 g <0.10 2.2 2.8 2.8 C18:4Stearidonic acid (ome3) g/100 g 0.23 0.3 0.22 0.3 C20:0 Arachidic acidg/100 g 0.43 0.35 0.29 0.34 C20:1 Eicosenoic acid g/100 g 1.1 1.4 1.31.2 C20:2 Eicosadienoic acid (om6) g/100 g 0.34 0.54 0.5 0.55C20:3Eicosatrienoic ac (omega6) g/100 g 0.14 0.24 0 0 C20:4 Arachidonicacid (omega6) g/100 g 0.78 1 0.18 0.27 C20:5Eicosapentaenoic ac (ome3)g/100 g 2.8 4.8 3.5 4.5 C21:0 Heneicosanoic acid g/100 g <0.10 0 0 0C22:0 Behenic acid g/100 g 1.3 0.58 0.44 0.55 C22:1 Erucic acid g/100 g<0.10 0.1 0.15 0.089 C22:2 Docosadienoic ac (ome6) g/100 g <0.10 00.0062 0 C22:5 Docosapentaenoic ac (ome3 g/100 g 2.3 3.8 2.8 3.6 C22:6Docosahexaenoic ac (ome3) g/100 g <0.10 4.5 3.9 4.5 C24:0 Lignocericacid g/100 g <0.10 0.11 0.13 0.073 C24:1 Nervonic acid g/100 g 2.6 0.180.14 0.11There are individual fatty acids showing differing values in some of thebatches, though the analysis shows a general uniformity in the fattyacid content between all batches.

DNA, RNA and Protein Analysis

Salmon roe have been homogenized using several homogenizing methods,which include centrifugation, separator, hydro press and milling. Usingthese different homogenizing methods it was important to confirm thestability of the product with regard to DNA, RNA and protein content. Toconfirm the general uniformity of the batches, an analysis was doneusing Nanodrop 2000c spectrophotometer. The resulting values have beenfilled in Table 15.

TABLE 15 Summary of measurements RNA, DNA and protein content inextracts Source LEX/corresp mg/ml μg/ml mg/ml of eggs Centrifugationspeed to LEX RNA DNA protein Salmon Homogenate, no LEX20 3.51 66.8 256centrifugation Salmon 15000 × g LEX20 2.34 44 252 Salmon Homogenate, noLEX24 3.42 192.4 180 centrifugation Salmon 12000 × g LEX24 2.93 50.8 208Trout Homogenate, no LEX28 2.67 131.6 249 centrifugation Trout 15000 × gLEX28 3.51 73.2 249 Trout Homogenate, no LEX25 2.53 528 296centrifugation Trout 15000 × g LEX25 3.70 72.8 262 Trout 15000 × g LEX253.63 99.2 210 Trout 12000 × g LEX31 4.59 87.2 270 Trout 12000 × g LEX324.68 124.8 — Trout 12000 × g LEX33 4.67 94.4 252 Salmon 12000 × g LEX35— — 192 Salmon Hydropress/separator LEX46 4.3 5.3 213 SalmonHydropress/separator LEX47 3.6 4.6 179 Salmon Mill/separator LEX48 4.15.1 201By comparing DNA and protein content in LEX batches produced withseveral production methods, values show slight differences betweenbatches and homogenization methods. However, the RNA content decreasesdramatically by the use of separator as a production method. Showingthat RNA may be precipitated or separated from the extract during theseparation step.

What is claimed is:
 1. A method of treating a subject comprising:contacting the skin of said subject with a water soluble, cytoplasmicfraction of salmonid eggs in an amount effective to cause one or moreeffects selected from the group consisting of reduction of fine lines inthe skin, normalization of skin color, increasing skin water content andhydration, decreasing or normalizing the amount of sebum in the skin,decreasing production of melanin, increasing collagen proteinproduction, increasing collagen gene expression, increasing adult stemcell proliferation, increasing cellular metabolism of carbohydrates,increasing cellular metabolism of lipids, prevention of apoptosis,increasing angiogenesis, upregulation the cell cycle of cells,increasing angiogenesis, increasing the hair cycle, increasingfollicular development, and increasing cell proliferation.
 2. The methodof claim 1, wherein said normalization of skin color comprises areduction of skin melanin index.
 3. The method of claim 1, wherein saidnormalization of skin color comprises a reduction of skin erythemaindex.
 4. The method of claim 1, wherein said water soluble, cytoplasmicfraction of salmonid eggs is provided in a cream, gel, emulsion,ointment, spray, powder or lotion.
 5. The method of claim 1, whereinsaid effect is reduction of fine lines in the skin.
 6. The method ofclaim 1, wherein said effect is normalization of skin color.
 7. Themethod of claim 1, wherein said effect is increasing skin water contentand hydration.
 8. The method of claim 1, wherein said effect isdecreasing or normalizing the amount of sebum in the skin.
 9. The methodof claim 1, wherein said effect is decreasing production of melanin. 10.The method of claim 1, wherein said effect is increasing collagenprotein production.
 11. The method of claim 1, wherein said effect isincreasing collagen gene expression.
 12. The method of claim 1, whereinsaid effect is increasing adult stem cell proliferation.
 13. The methodof claim 1, wherein said effect is increasing cellular metabolism ofcarbohydrates.
 14. The method of claim 1, wherein said effect isincreasing cellular metabolism of lipids.
 15. The method of claim 1,wherein said effect is prevention of apoptosis.
 16. The method of claim1, wherein said effect is increasing angiogenesis.
 17. The method ofclaim 1, wherein said effect is upregulation the cell cycle of cells.18. The method of claim 1, wherein said effect is increasing the haircycle.
 19. The method of claim 1, wherein said effect is increasingfollicular development.
 20. The method of claim 1, wherein said effectis increasing cell proliferation.